Gelsolin binding agent compositions and uses of same

ABSTRACT

The invention relates generally to gelsolin binding agents (e.g., antibodies) which can bind to gelsolin polypeptides. Gelsolin binding agents of the invention are useful, alone or in combination, to detect a gelsolin polypeptide (a.k.a., the target polypeptide) in a test sample as well as to purify native gelsolin proteins. Gelsolin binding agents are also useful to diagnose, a gelsolin related medical condition in subjects in need thereof. Kits to detect gelsolin in biological samples are provided by the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority as a national stage applicationof International Application No. PCT/CN2007/002467 filed on Aug. 15,2007, the entire contents of which are incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

This invention relates generally to the preparation of gelsolin bindingagents and uses of the same. In particular, the present inventionrelates to the preparation of anti-gelsolin antibodies that recognize anantigen determinant (i.e., epitope) of human plasma gelsolin and theiruse for gelsolin detection.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Actin is the most abundant protein in animal cells and constitutes10-20% of the protein of many nucleated cells and 30% of the protein ofmuscle cells. Actin molecules each bind an ATP molecule andself-assemble into long filaments during which the ATP is hydrolyzedinto ADP.

Injury to animal tissues results in the release of actin into theextracellular space, including the bloodstream. Although approximatelyhalf of nonmuscle cell actin is F-actin, (the double-helical, rodlike,filament form of actin which is assembled from G-actin monomers), theionic conditions of extracellular fluids favor actin polymerization, sothat virtually all the actin released into the blood from dying cellswould be expected to polymerize into filaments (Lind, S. E. et al., Am.Rev. Respir. Dis. 138:429-434 (1988)). In purified solutions, in theabsence of filament-shortening proteins, actin filaments can easilyattain lengths of several microns. Were some fraction of actin releasedfrom injured cells to be irreversibly denatured, however, or else boundto one of the intracellular actin-binding proteins discussed below, thisactin would remain monomeric.

There are many proteins which naturally associate with actin (for areview of actin-binding proteins, see Stossel et al., Ann. Rev. CellBiol. 1: 353-402 (1985); Pollard et al., Ann. Rev. Biochem. 55:987-1035(1986)). However, two proteins, gelsolin and DBP (vitamin D bindingprotein) are thought to be primarily responsible for bindingextracellular actin. (Janmey et al., Blood 70:529-530 (1987)). Gelsolinis an actin-binding protein that is a key regulator of actin filamentassembly and disassembly. Gelsolin is an 82-kDa protein with sixhomologous subdomains, referred to as S1-S6. Each subdomain is composedof a five-stranded β-sheet, flanked by two α-helices, one positionedperpendicular with respect to the strands and one positioned parallel.The N-terminal (S1-S3) forms an extended β-sheet, as does the C-terminal(S4-S6) (Kiselar et al. PNAS 100: 3942-3947 (2003)). The protein ishighly conserved and highly homologous among species. Gelsolin islocated intracellularly (in cytosol and mitochondria) andextracellularly (in blood plasma). Koya et al., J Biol Chem 275 (20):15343-15349 (2000).

Gelsolin has several functions in regulating actin polymerization.First, gelsolin is involved in monomeric actin binding. In the presenceof Ca²⁺, gelsolin binds two actin monomers. Gelsolin can also bind actinfilaments by a another actin binding site. Second, gelsolin binds twoactin monomers to form a nucleus for actin polymerization and caps thebarbed end of actin filaments. Thus, gelsolin is capable of both servingas a nucleus for actin polymerization and capping the ends of thenascent microfilaments. Finally, gelsolin has actin severing activity.

Because of the large amounts of actin in cells, the release of actinfrom dying cells provides sufficient actin to have a significant affecton the microenvironment, either by increasing the viscosity ofextracellular fluids of plasma and/or by entrapping cells or by other,as yet unidentified toxic effects. Infusion of extracellular free actinis toxic to animal tissues, and especially to renal and cardiopulmonarysystems (Harper et al., Clin. Res. 36:625 A (1988); Haddad et al., PNAS87: 1381-1385 (1990)). Acute renal failure is a complication of muscleinjury and actin infusions in rats causes transient elevations of theblood urea nitrogen (BUN) and creatinine levels, consistent with renalfailure. Free actin in the plasma may form filaments which may lead tomultiple organ dysfunction syndrome (Dahl et al., Shock 12(2): 102-4(1999)). Moreover, since each extracellular actin molecule in a filamenthas an ADP molecule associated with it, the presence of extracellularactin in the blood may tend to induce or augment platelet aggregation ina manner which may not be advantageous to the host (Lind et al., Am.Rev. Respir. Dis. 138:429-434 (1988); Scarborough et al., Biochem.Biophy. Res. Commun. 100:1314-1319 (1981)). Consequently, plasmagelsolin has a vital function of scavenging actin released from dead anddying cells and plasma gelsolin levels appear to be an early prognosticmarker in patients experiencing trauma (Mounzer et al., Am. J. Respir.Crit. Care Med. 160: 1673-81 (1999)).

SUMMARY OF THE INVENTION

This invention relates generally to the preparation of gelsolin bindingagents and uses of the same. In particular, the present inventionrelates to the preparation of anti-gelsolin antibodies that recognize anantigen determinant (i.e., epitope) of human plasma gelsolin and theiruse for gelsolin detection. In one aspect, the invention provides anantibody or antigen-binding fragment thereof having the sameantigen-binding specificity of antibodies produced by a deposited cellline selected from the group consisting of CGMCC Accession Nos: 2114,2115, and 2116. In one embodiment, the invention provides, an antibodyor an antigen-binding fragment thereof, comprising at least heavy chainCDR3 amino acid sequence selected from the group consisting of:FAQGALKSED (SEQ ID NO.: 2), SEPDGFWEAL (SEQ ID NO.: 3), and ACSNKIGRFV(SEQ ID NO.: 4) or a variant thereof having one or more conservativeamino acid substitutions, wherein the antibody or the fragment thereofspecifically binds gelsolin. In one embodiment, the invention providesnucleic acids compositions encoding an antibody or an antigen-bindingfragments of the invention. In one embodiment, the invention provides avector comprising nucleic acids composition encoding an antibody or anantigen-binding fragment of the invention. The vector may furthercomprise a promoter operably-linked to the nucleic acid molecule. In oneembodiment, the invention provides a host cell that comprises a vectorcomprising nucleic acids composition encoding an antibody or anantigen-binding fragment of the invention. In one embodiment, theinvention provides a continuous cell line which produces a monoclonalantibody, wherein the monoclonal antibody binds to the same antigenicdeterminant as an antibody produced by a hybridoma cell line selectedfrom the group consisting of: CGMCC Accession Nos: 2114, 2115, and 2116,wherein the cell line is produced by the process of fusing a lymphocytederived from a mouse immunized with carcinoma cells or an immunogenicdeterminant thereof and a mouse myeloma cell.

In another aspect, the invention provides a method for preparing anantibody or fragment thereof that binds immuno specifically to apolypeptide of SEQ ID NO.:1, the method comprising the steps of: (a)culturing a cell containing a nucleic acid according to claim 4 underconditions that provide for expression of the antibody or fragmentthereof; and (b) recovering the expressed antibody or fragment thereof.

In another aspect, the invention provides an isolated epitope ofgelsolin comprising an amino acid sequence selected from the groupconsisting of: FAQGALKSED (SEQ ID NO.: 2), SEPDGFWEAL (SEQ ID NO.: 3),and ACSNKIGRFV (SEQ ID NO.: 4), wherein the epitope is recognized by anantibody capable of binding full-length human gelsolin. In oneembodiment, invention provides an antibody or an antigen-bindingfragment thereof generated by preparation of an immunogen containing anepitope of gelsolin comprising an amino acid sequence selected from thegroup consisting of: FAQGALKSED (SEQ ID NO.: 2), SEPDGFWEAL (SEQ ID NO.:3), and ACSNKIGRFV (SEQ ID NO.: 4).

In one aspect, the invention provides a method for determining thepresence or amount of gelsolin in a biological sample comprising thesteps of: (a) contacting a biological sample with one or more of theantibodies or antigen-binding fragments thereof having the sameantigen-binding specificity produced by a deposited cell line selectedfrom the group consisting of: CGMCC Accession Nos: 2114, 2115, and 2116under conditions wherein the antibody or fragment thereof specificallybinds to gelsol in; and (b) detecting the presence or amount of antibodyor fragment thereof bound to the gelsolin, thereby determining thepresence or amount of the gelsolin in the sample. In one embodiment ofthe method, the sample is contacted with said antibody or anantigen-binding fragment in an ELISA. In one embodiment of the method,the step of contacting comprises binding a first antibody to a substrateand contacting the sample and binding a second antibody to thesubstrate, wherein the second antibody comprises a detectable label. Inone embodiment of the method, the first antibody binds to the sameantigenic determinant as an antibody produced by a hybridoma cell lineCGMCC Accession No: 2115 and the second antibody binds to the sameantigenic determinant as an antibody produced by a hybridoma cell lineselected from the group consisting of CGMCC Accession No. 2114 and 2116.

In one aspect, the invention provides a method for monitoring septicshock in a mammalian subject, the method comprising the steps of: (a)measuring the level of gelsolin in a mammalian subject according to amethod of the invention for determining the presence or amount ofgelsolin in a biological sample; (b) comparing the level of gelsolin inthe first subject to a reference standard, wherein the referencestandard comprises a control subject not having septic shock, andwherein a decrease in the level of gelsolin of the first subjectcompared to the reference standard indicates that the mammalian subjecthas septic shock.

In another aspect, the invention provides a method of selecting amammalian subject for inclusion in a clinical trial for determining theefficacy of a compound to prevent or treat a medical condition,comprising the steps of: (a) measuring the level of gelsolin in themammalian subject according to a method of the invention for determiningthe presence or amount of gelsolin in a biological sample; (b) comparingthe level of gelsolin in the first subject to a reference standard,wherein the reference standard comprises a control mammalian subject nothaving a disease or condition affecting gelsolin levels, and (c)selecting to include the mammalian subject in the clinical trial,wherein a similarity in the gelsolin level of the mammalian subject issimilar to the gelsolin level of the reference standard.

In another aspect, the invention provides a method for determining thepresence of, or predisposition to, a disease or condition associatedwith altered levels of a gelsolin polypeptide in a first mammaliansubject, the method comprising the steps of: (a) providing a test samplefrom the first mammalian subject; (b) contacting the test sample fromthe first mammalian subject with one or more compounds that bind thegelsolin polypeptide to form a compound/gelsolin polypeptide complex,wherein the compound is an antibody or antigen-binding fragment thereofhaving the same antigen-binding specificity produced by a hybridoma cellline selected from the group consisting of CGMCC Accession No. 2114,2115, and 2116; (c) detecting the level of compound/gelsolin polypeptidecomplex; (d) quantifying the level of expression of the gelsolinpolypeptide in the sample from the first mammalian subject; and (e)comparing the amount of the gelsolin polypeptide in the sample of step(a) to the amount of polypeptide present in a control sample from asecond mammalian subject known not to have, or not to be predisposed to,the disease or condition, wherein an alteration in the expression levelof the gelsolin polypeptide in the first subject as compared to thecontrol sample indicates the presence of, or predisposition to, thedisease or condition. In one embodiment of the method, sample iscontacted with the compound in an ELISA. In one embodiment of themethod, the step of contacting comprises binding a first antibody to asubstrate and contacting the sample and a second antibody to thesubstrate, wherein the second antibody comprises a detectable label. Inone embodiment of the method, the first antibody binds to the sameantigenic determinant as an antibody produced by a hybridoma cell lineCGMCC Accession No: 2115 and the second antibody binds to the sameantigenic determinant as an antibody produced by a hybridoma cell lineselected from the group consisting of CGMCC Accession No. 2114 and 2116.In one embodiment of the method, the disease or condition associatedwith altered levels of gelsolin is selected from the group consistingof: septic shock, multiple organ dysfunction syndrome rheumatoidarthritis, stroke, heart infarction, cancer, systemic autoimmunedisease, chronic hepatitis, side-effects of chemotherapy, andside-effects of radiation therapy.

In one aspect, the invention provides a method for selecting aprophylactic or therapeutic treatment for a subject, comprising thesteps of: (a) measuring the level of gelsolin in the mammalian subjectaccording to a method of the invention for determining the presence oramount of gelsolin in a biological sample; (b) assigning the subject toa subject class based on the level of gelsolin of the subject; and (c)selecting a prophylactic or therapeutic treatment based on the subjectclass.

In one aspect, the invention provides a method of purifying gelsolin,the method comprising the steps of (a) contacting a biological samplecomprising gelsolin with at least one immobilized antibody orantigen-binding fragment thereof to form an immobilized gelsolinantibody complex under conditions wherein the antibody or fragmentthereof specifically binds to gelsolin, wherein the antibodies orantigen-binding fragments thereof have the same antigen-bindingspecificity produced by a deposited cell line selected from the groupconsisting of: CGMCC Accession Nos: 2114, 2115, and 2116; and (b)recovering the gelsolin from the immobilized gelsolin antibody complex.In one embodiment of the method, the biological sample comprises humanserum.

In one aspect, the invention provides a kit comprising one or morecontainers, one or more antibodies, or antigen-binding fragmentsthereof, having the same antigen-binding specificity produced by adeposited cell line selected from the group consisting of: CGMCCAccession Nos: 2114, 2115, and 2116 and instructions for using thecontents therein.

In one aspect, the invention provides a kit comprising: (a) an ELISAplate coated with a first antibody; and (b) a second antibody in acontainer, wherein the first and second antibodies are antibodiesproduced by a deposited cell line selected from the group consisting of:CGMCC Accession Nos: 2114, 2115, and 2116. In one embodiment, the kitfurther comprising one or more of the following components: a humanplasma gelsolin standard, human plasma dilution buffer, washing buffer,and substrate buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a SDS-PAGE analysis of human plasma gelsolin proteins used asimmunogens for generation of mouse anti-human gelsolin monoclonalantibodies: native human plasma gelsolin (Lane 2), and recombinantfull-length gelsolin (Lane 3), a recombinant N-terminal gelsolinfragment (Lane 4), and a recombinant C-terminal gelsolin fragment (Lane5). A molecular weight marker is shown in Lane 1.

FIG. 2 is an ELISA analysis of the binding charateriscs of anti-gelsolinmonoclonal antibodies to different forms of human gelsolin (NG: nativegelsolin; GF: full-length recombinant gelsolin; GN: recombinantN-terminal gelsolin fragment; GC: recombinant C-terminal gelsolinfragment) and BSA control. Each panel presents data from a differentantibody: Panel A. GN3E9 (FIG. 2A); Panel B. GF2D6 (FIG. 2B); Panel C.GC1C10 (FIG. 2C); and Panel D. GS2C4 (FIG. 2D).

FIG. 3 is a western blot analysis of SDS-PAGE of two human plasmasamples probed with anti-gelsolin monoclonal antibodies.

FIG. 4 is a SDS-PAGE analysis of human plasma gelsolinimmunoprecipitated with anti-gelsolin antibodies. The samples in eachlane were as follows: lane 1: blank control (no plasma); lane 2: GC1C10;lane 3: GF2D6; lane 4 GN3E9; and lane 5: GS2C4.

FIG. 5 is an analysis of the cross-reactivity of anti-gelsolinantibodies, GC 1C10 (FIG. 5A); GF2D6 (FIG. 5B) and GN3E9 (FIG. 5C) byimmunoprecipitation (top panel) and western blot analysis (lower panel)among seven different species: lane 1: mouse; lane 2: monkey; lane 3:rabbit; lane 4: rat; lane 5: bovine; lane 6: horse; lane 7: human.

FIG. 6 is a 3D structure of the C-terminal fragment of gelsolin and theepitope location of anti-gelsolin antibodies.

FIG. 7 is a F-actin inihibitory ELISA analysis of the binding ofanti-gelsolin antibodies to purified human plasma gelsolin. Theinhibition of antibody binding to gelsolin by F-actin is presented aspercent of the binding without F-actin.

FIG. 8 is a standard curve of human plasma gelsolin generated by achemiluminescence ELISA using a pair of antibodies, GN3E9/GC1C10 (FIG.8A) or GN3E9/GF2D6 (FIG. 8B), showing plasma gelsolin concentration(ng/mL) as a function of detection signal (relative light units, RLU).

FIG. 9 is a comparison of quantification of plasma gelsolin levels basedsample handling conditions, i.e. addition of sodium citrate (citrate),heparin, or EDTA to the sample.

FIG. 10 shows a graph of optical density versus gelsolin polypeptideconcentration in an ELISA assay using the antibody pair GN3E9/GC1C10,where the gelsolin polypeptide is NG: native gelsolin; GF: full-lengthrecombinant gelsolin; GN: recombinant N-terminal gelsolin fragment; orGC: recombinant C-terminal gelsolin fragment.

FIG. 11 shows a graph of optical density versus plasma gelsolin in anELISA assay using the GN3E9/GC 1C10 (FIG. 11A) or GN3E9/GF2D6 (FIG. 11B)antibody pair to measure actin-free gelsolin (control) or gelsolin incomplex with actin (+actin).

FIG. 12 is a graph of serum gelsolin concentration measured in healthycontrols (n=291) and ICU (critical care) patients (n=22) using agelsolin ELISA assay of the invention.

FIG. 13 is a graph of serum gelsolin concentration (μg/mL) in normalpatients and patients exhibiting inactive and active forms of SystemicLupus Erythematosus (SLE) as determined using a gelsolin ELISA assay ofthe invention.

FIG. 14 is a graph of serum gelsolin concentration (μg/mL) in normalpatients and patients with chronic hepatitis as determined using agelsolin ELISA assay of the invention.

FIG. 15 is a western blot analysis of the effect of chemotherapy onplasma gelsolin levels. FIG. 15A is a photograph of western blot andFIG. 15B is a quantitative analysis of the western blot by densitometry.

FIG. 16 is a western blot analysis of the time-dependent depletion ofplasma gelsolin after chemotherapy. FIG. 16A is a photograph of awestern blot of serum samples of mice at various time points afterreceiving adriamycin. FIG. 16B is a quantitative analysis of the westernblot of FIG. 16A by densitometry. FIG. 16C is a photograph of a westernblot of serum samples of mice at various time points after receivingTaxol. FIG. 16D is a quantitative analysis of the western blot in FIG.16C by densitometry.

FIG. 17 is a graph showing plasma gelsolin levels of five patients inchemotherapy, a time-dependent decrease in a group of patients withovarian cancer after cisplatin chemotherapy as determined using agelsolin ELISA assay of the invention.

FIG. 18 is a graph showing the therapeutic efficacy of full-lengthrecombinant gelsolin in reduction of chemotherapy-induced body weightloss (FIG. 18A) and mortility (FIG. 18B) in an in vivo murine model.

FIG. 19 is a SDS-PAGE of affinity-purified human plasma gelsolin by theGC1C10, GN3E9, and GC2D6 antibodies of the invention.

FIG. 20 is a western blot analysis of different forms of human plasmagelsolin with three representative anti-gelsolin antibodies. Panel A isa representative of a group of antibodies that only react with 90 kDaform of human plasma gelsolin (FIG. 20A). Panel B is a representative ofa group of antibodies that only reacts with the 50 kDa gelsolin-likepolypeptide (FIG. 20B). Panel C is representative of a group ofantibodies that reacts with both 90 kDa and 50 kDa immunoreactive forms(FIG. 20C).

FIG. 21 is a western blot analysis of intracellular gelsolin of a humanpancreatic cancer cell line with or without apoptosis with anti-gelsolinantibodies. Human pancreatic cancer cells, MIAcapa, cultured in controlmedium (lane 1) or in the presence of 1000 ng/ml of anti-DR5 antibody(CTB006) for four hours (lane 2). The western blot of total cell lysateswas probed with a gelsolin C-terminal fragment specific antibody GC1C10(FIG. 21A) or a gelsolin N-terminal fragment specific antibody GN3E9(FIG. 21B).

FIG. 22 is an SDS-PAGE analysis of immunoprecipitated plasma gelsolin(top panel) and a western blot analysis (lower panel) of total plasmagelsolin in a first study of 4 healthy control patients (N1 to N4) and 4patients with cancer (C1 to C4) (FIG. 22A) and a second study of 4healthy control patients (N5 to N8) and 4 patients with cancer (C5 toC8) (FIG. 22B).

FIG. 23 is a graph of serum gelsolin concentration (μg/mL) in normalpatients compared with patients with rheumatoid arthritis as determinedusing a gelsolin ELISA assay of the invention.

DETAILED DESCRIPTION

General. It is to be appreciated that certain aspects, modes,embodiments, variations and features of the invention are describedbelow in various levels of detail in order to provide a substantialunderstanding of the present invention.

The invention generally provides gelsolin binding agents (e.g.,antibodies) which can bind to gelsolin polypeptides. Accordingly, thevarious aspects of the present invention relate to the preparation,expression and characterization of gelsolin binding agents. Gelsolinbinding agents of the invention are useful, alone or in combination, todetect a gelsolin polypeptide (a.k.a., the target polypeptide) in a testsample as well as in methods to purify native gelsolin proteins,including native gelsolin polypeptides from a biological sample.Gelsolin binding agents are useful to diagnose a gelsolin-relatedmedical condition in subjects in need thereof. An amino acid sequence ofa human gelsolin polypeptide (SEQ ID NO.: 1) is shown in Table 1.

TABLE 1 Human Gelsolin Polypeptide Sequence (SEQ ID NO.: 1)MAPHRPAPALLCALSLALCALSLPVRAATASRGASQAGAPQGRVPEARPNSMVVEHPEFLKAGKEPGLQIWRVEKFDLVPVPTNLYGDFFTGDAYVILKTVQLRNGNLQYDLHYWLGNECSQDESGAAAIFTVQLDDYLNGRAVQHREVQGFESATFLGYFKSGLKYKKGGVASGFKHVVPNEVVVQRLFQVKGRRVVRATEVPVSWESFNNGDCFILDLGNNIHQWCGSNSNRYERLKATQVSKGIRDNERSGRARVHVSEEGTEPEAMLQVLGPKPALPAGTEDTAKEDAANRKLAKLYKVSNGAGTMSVSLVADENPFAQGALKSEDCFILDHGKDGKIFVWKGKQANTEERKAALKTASDFITKMDYPKQTQVSVLPEGGETPLFKQFFKNWRDPDQTDGLGLSYLSSHIANVERVPFDAATLHTSTAMAAQHGMDDDGTGQKQIWRIEGSNKVPVDPATYGQFYGGDSYIILYNYRHGGRQGQIIYNWQGAQSTQDEVAASAILTAQLDEELGGTPVQSRVVQGKEPAHLMSLFGGKPMIIYKGGTSREGGQTAPASTRLFQVRANSAGATRAVEVLPKAGALNSNDAFVLKTPSAAYLWVGTGASEAEKTGAQELLRVLRAQPVQVAEGSEPDGFWEALGGKAAYRTSPRLKDKKMDAHPPRLFACSNKIGRFVIEEVPGELMQEDLATDDVMLLDTWDQVFVWVGKDSQEEEKTEALTSAKRYIETDPANRDRRTPITVVKQGFEPPSFVGWFLGWDDDYWSVDPLDRAMAELAA

In some embodiments, the gelsolin binding agents (e.g. anti-gelsolin oranti-gelsolin like antibodies) of the present invention detect theactive, or unbound, form of gelsolin. While not wishing to be limited bytheory, free and complexed (to actin) gelsolin molecules differ in theirfunctional properties. Although free gelsolin can sever actin filaments,actin-gelsol in complexes cannot. Gelsolin's severing activity isactivated by micromolar Ca²⁺ and has been shown to be inhibited byphosphatidyl inositol bisphosphate (PIP₂) and phosphatidyl inositolmonophosphate (PIP). Since extracellular Ca²⁺ concentrations are atmillimolar levels and extracellular fluids do not normally contain PIPor PIP₂ in a form that inhibits gelsolin, plasma gelsolin isconstitutively active in extracellular fluids.

The various aspects of the present invention further relate todiagnostic methods and kits that use the gelsolin binding agents of theinvention to identify individuals predisposed to a medical condition orto classify individuals with regard to drug responsiveness, sideeffects, or optimal drug dose. In other aspects, the invention providesmethods for purifying gelsolin or gelsolin-like polypeptides from abiological sample, including, for example, native human gelsolin fromplasma. Accordingly, various particular embodiments that illustratethese aspects follow.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and the claims.Generally, enzymatic reactions and purification steps are performedaccording to the manufacturer's specifications.

In practicing the present invention, many conventional techniques inmolecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used.

These techniques are well-known and are explained in, e.g., CurrentProtocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997);Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989);DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985);Oligonuchotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization,Hames & Higgins, Eds. (1985); Transcription and Translation, Hames &Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986);Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A PracticalGuide to Molecular Cloning; the series, Meth. Enzymol., (Academic Press,Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos,Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. Enzymol.,Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively. Methods todetect and measure levels of polypeptide gene expression products (i.e.,gene translation level) are well-known in the art and include the usepolypeptide detection methods such as antibody detection andquantification techniques. (See also, Strachan & Read, Human MolecularGenetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. As used inthis specification and the appended claims, the singular forms “a”, “an”and “the” include plural referents unless the content clearly dictatesotherwise. For example, reference to “a cell” includes a combination oftwo or more cells, and the like. Generally, the nomenclature used hereinand the laboratory procedures in cell culture, molecular genetics,organic chemistry, analytical chemistry and nucleic acid chemistry andhybridization described below are those well known and commonly employedin the art. All references cited herein are incorporated herein byreference in their entireties and for all purposes to the same extent asif each individual publication, patent, or patent application wasspecifically and individually incorporated by reference in its entiretyfor all purposes.

Definitions. The definitions of certain terms as used in thisspecification are provided below. Definitions of other terms may befound in the Illustrated Dictionary of Immunology, 2nd Edition (Cruse,J. M. and Lewis, R. E., Eds., Boca Raton, Fla.: CRC Press, 1995). Unlessindicated otherwise, the term “gelsolin” when used herein refer to humanprotein and gene.

As used herein, the “administration” of an agent or drug to a subject orsubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),rectally, or topically. Administration includes self-administration andthe administration by another. It is also to be appreciated that thevarious modes of treatment or prevention of medical conditions asdescribed are intended to mean “substantial”, which includes total butalso less than total treatment or prevention, and wherein somebiologically or medically relevant result is achieved.

As used herein, the term “amino acid” includes naturally-occurring aminoacids and synthetic amino acids, as well as amino acid analogs and aminoacid mimetics that function in a manner similar to thenaturally-occurring amino acids. Naturally-occurring amino acids arethose encoded by the genetic code, as well as those amino acids that arelater modified, e.g., hydroxyproline, γ-carboxyglutamate, andO-phosphoserine. Amino acid analogs refers to compounds that have thesame basic chemical structure as a naturally-occurring amino acid, i.e.,an α-carbon that is bound to a hydrogen, a carboxyl group, an aminogroup, and an R group, e.g., homoserine, norleucine, methioninesulfoxide, methionine methyl sulfonium. Such analogs have modified Rgroups (e.g., norleucine) or modified peptide backbones, but retain thesame basic chemical structure as a naturally-occurring amino acid Aminoacid mimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally-occurring amino acid. Aminoacids can be referred to herein by either their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, can bereferred to by their commonly accepted single-letter codes.

As used herein, the term “antibody” means a polypeptide comprising aframework region from an immunoglobulin gene or fragments thereof thatspecifically binds and recognizes an antigen, e.g., a gelsolinpolypeptide. Use of the term antibody is meant to include wholeantibodies, including single-chain whole antibodies, and antigen-bindingfragments thereof. The term “antibody” includes bispecific antibodiesand multispecific antibodies so long as they exhibit the desiredbiological activity or function.

As used herein, the term “antibody-related polypeptide” meansantigen-binding antibody fragments, including single-chain antibodies,that can comprise the variable region(s) alone, or in combination, withall or part of the following polypeptide elements: hinge region, CH₁,CH₂, and CH₃ domains of an antibody molecule. Also included in theinvention are any combinations of variable region(s) and hinge region,CH₁, CH₂, and CH₃ domains. Antibody-related molecules useful as bindingagents of the invention include, e.g., but are not limited to, Fab, Fab′and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain. Examples include: (i) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and CH₁ domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the V_(H) and CH₁ domains; (iv) a Fv fragment consistingof the V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAbfragment (Ward et al., Nature 341: 544-546, 1989), which consists of aV_(H) domain; and (vi) an isolated complementarity determining region(CDR). As such “antibody fragments” can comprise a portion of a fulllength antibody, generally the antigen binding or variable regionthereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.Single-chain antibody molecules may comprise a polymer with a number ofindividual molecules, for example, dimmer, trimer or other polymers.

As used herein, the term “biological sample” means sample materialderived from or contacted by living cells. The term “biological sample”is intended to include tissues, cells and biological fluids isolatedfrom a subject, as well as tissues, cells and fluids present within asubject. Biological samples of the invention include, e.g., but are notlimited to, whole blood, plasma, semen, saliva, tears, urine, fecalmaterial, sweat, buccal, skin, cerebrospinal fluid, and hair. Biologicalsamples can also be obtained from biopsies of internal organs or fromcancers. Biological samples can be obtained from subjects for diagnosisor research or can be obtained from undiseased individuals, as controlsor for basic research.

As used herein, the term “CDR-grafted antibody” means an antibody inwhich at least one CDR of an “acceptor” antibody is replaced by a CDR“graft” from a “donor” antibody possessing a desirable antigenspecificity.

As used herein, the term “chimeric antibody” means an antibody in whichthe Fc constant region of a monoclonal antibody from one species (e.g.,a mouse Fc constant region) is replaced, using recombinant DNAtechniques, with an Fc constant region from an antibody of anotherspecies (e.g., a human Fc constant region). See generally, Robinson etal., PCT/US86/02269; Akira et al., European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European PatentApplication 125,023; Better et al., Science 240: 1041-1043, 1988; Liu etal., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J.Immunol. 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Woodet al., Nature 314: 446-449, 1885; and Shaw et al., J. Natl. CancerInst. 80: 1553-1559, 1988.

As used herein, the term “clinical response” means any or all of thefollowing: a quantitative measure of the response, no response, andadverse response (i.e., side effects).

As used here, in the term “clinical trial” means any research studydesigned to collect clinical data on responses to a particulartreatment, and includes, but is not limited to phase I, phase II, andphase III clinical trials. Standard methods are used to define thepatient population and to enroll subjects.

As used herein, the term “consensus FR” means a framework (FR) antibodyregion in a consensus immunoglobulin sequence. The FR regions of anantibody do not contact the antigen.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) in the same polypeptide chain (V_(H) V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen binding sites. Diabodies aredescribed more fully in, e.g., EP 404,097; WO 93/11161; and 30 Hollingeret al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

As used herein, the term “effector cell” means an immune cell which isinvolved in the effector phase of an immune response, as opposed to thecognitive and activation phases of an immune response. Exemplary immunecells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes(e.g., B cells and T cells including cytolytic T cells (CTLs)), killercells, natural killer cells, macrophages, monocytes, eosinophils,neutrophils, polymorphonuclear cells, granulocytes, mast cells, andbasophils. Effector cells express specific Fc receptors and carry outspecific immune functions. An effector cell can induceantibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophilcapable of inducing ADCC. For example, monocytes, macrophages,neutrophils, eosinophils, and lymphocytes which express FcαR areinvolved in specific killing of target cells and presenting antigens toother components of the immune system, or binding to cells that presentantigens. An effector cell can also phagocytose a target antigen, targetcell, metastatic cancer cell, or microorganism.

As used herein, the term “epitope” means a protein determinant capableof specific binding to an antibody. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. In one embodiment, an “epitope” of gelsolin is aregion in the gelsolin protein to which the gelsolin binding agent ofthe invention binds. In select embodiments of the invention, thisepitope is in the domain spanning amino acid residues from about 321 toabout 330, from about 636 to about 645, or from about 661 to 670 of SEQID NO.: 1.

To screen for gelsolin binding agents which bind to an epitope, aroutine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. This assay can be used to determine if agelsolin binding agent binds the same site or epitope as a gelsolinantibody of the invention. Alternatively, or additionally, epitopemapping can be performed by methods known in the art. For example, theantibody sequence can be mutagenized such as by alanine scanning, toidentify contact residues. In a different method, peptides correspondingto different regions of gelsolin can be used in competition assays withthe test antibodies or with a test antibody and an antibody with acharacterized or known epitope.

As used herein, the term “effective amount” or “pharmaceuticallyeffective amount” or “therapeutically effective amount” of acomposition, is a quantity sufficient to achieve a desired therapeuticand/or prophylactic effect, e.g., an amount which results in theprevention of, or a decrease in, the symptoms associated with a diseasethat is being treated, e.g., the diseases or medical conditionsassociated with target polypeptide (e.g. gelsolin or gelsolin-likepolypeptides). The amount of a composition of the invention administeredto the subject will depend on the type and severity of the disease andon the characteristics of the individual, such as general health, age,sex, body weight and tolerance to drugs. It will also depend on thedegree, severity and type of disease. The skilled artisan will be ableto determine appropriate dosages depending on these and other factors.The compositions of the present invention can also be administered incombination with one or more additional therapeutic compounds. In themethods of the present invention, gelsolin may be administered to asubject having decreased gelsolin levels caused by a disease ortraumatic condition, thereby increasing the level of plasma gelsolin inthe subject. For example, a “therapeutically effective amount” ofgelsolin is meant levels in which the toxic effects of freeextracellular actin are, at a minimum, ameliorated.

As used herein, “expression” includes but is not limited to one or moreof the following: transcription of the gene into precursor mRNA;splicing and other processing of the precursor mRNA to produce maturemRNA; mRNA stability; translation of the mature mRNA into protein(including codon usage and tRNA availability); and glycosylation and/orother modifications of the translation product, if required for properexpression and function.

As used herein, the term “gelsolin” refers to a multifunctional actinbinding protein. In mammals, gelsolin is comprises two isoforms:cytoplasmic and extracellular variants. Human plasma gelsolin differsfrom cellular gelsolin only by the addition of about 25 amino acids tothe N-terminus of the molecule and both gelsolins are the product of asingle gene. Plasma gelsolin has three actin-binding sites and bindswith high affinity to either G-actin or F-actin. “Gelsolin” also refersto recombinant forms of the mammalian polypeptide.

As used herein, the term “gene” means a segment of DNA that contains allthe information for the regulated biosynthesis of an RNA product,including promoters, exons, introns, and other untranslated regions thatcontrol expression.

As used herein, the term “human sequence antibody” includes antibodieshaving variable and constant regions (if present) derived from humangermline immunoglobulin sequences. The human sequence antibodies of theinvention can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo). Suchantibodies can be generated in non-human transgenic animals, e.g., asdescribed in PCT Publication Nos. WO 01/14424 and WO 00/37504. However,the term “human sequence antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (e.g., humanized antibodies).

As used herein, the term “humanized” forms of non-human (e.g., murine)antibodies are chimeric antibodies which contain minimal sequencederived from non-human immunoglobulin. For the most part, humanizedantibodies are human immunoglobulins in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance such asbinding affinity. Generally, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence although theFR regions may include one or more amino acid substitutions that improvebinding affinity. The number of these amino acid substitutions in the FRare typically no more than 6 in the H chain, and in the L chain, no morethan 3. The humanized antibody optionally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

“Amino acid sequence modification(s)” of the gelsolin antibodiesdescribed herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of a gelsolin antibody areprepared by introducing appropriate nucleotide changes into the antibodynucleic acid, or by peptide synthesis. Such modifications include, forexample, deletions from, and/or insertions into and/or substitutions of,residues within the amino acid sequences of the gelsolin antibody. Anycombination of deletion, insertion, and substitution is made to obtainthe antibody of interest, as long as the obtained antibody possesses thedesired properties. The modification also includes the change of thepattern of glycosylation of the protein. A useful method foridentification of preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells in Science,244: 1081-1085 (1989). The mutated antibody is then screened for thedesired activity, The invention includes antibody variants with one ormore amino acid addition, deletion and/or substitution of the amino acidsequence defined by hybridomas GC1C10, GN3E9, or GF2D6 having CGMCCAccession Numbers 2114, 2115, 2116, respectively, provided that theantibody variant possesses the desired properties.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the Y_(L), and aroundabout 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the V_(H) (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991))and/or those residues from a “hypervariable loop” (e.g. residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 52A-55(H2) and 96-101 (H3) in the V_(H) (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

As used herein, the terms “identical” or percent “identity”, when usedin the context of two or more nucleic acids or polypeptide sequences,refers to two or more sequences or subsequences that are the same orhave a specified percentage of amino acid residues or nucleotides thatare the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higheridentity over a specified region (e.g., nucleotide sequence encoding anantibody described herein or amino acid sequence of an antibodydescribed herein), when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site). Such sequences are then said to be “substantiallyidentical.” This term also refers to, or can be applied to, thecomplement of a test sequence. The term also includes sequences thathave deletions and/or additions, as well as those that havesubstitutions. As described below, the preferred algorithms can accountfor gaps and the like. Preferably, identity exists over a region that isat least about 25 amino acids or nucleotides in length, or morepreferably over a region that is 50-100 amino acids or nucleotides inlength.

An “isolated” or “purified” polypeptide or biologically-active portionthereof is substantially free of cellular material or othercontaminating polypeptides from the cell or tissue source from which thegelsolin binding agent is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. For example,an isolated gelsolin binding agent which is an anti-gelsolin oranti-gelsolin-like antibody would be free of materials that wouldinterfere with diagnostic or therapeutic uses of the agent. Suchinterfering materials may include enzymes, hormones and otherproteinaceous and nonproteinaceous solutes. Alternatively, an isolatedgelsolin or gelsolin-like polypeptide, which is immunoractive with agelsolin binding agent of the invention, would be substantially free ofmaterials that would interfere with diagnostic or therapeutic uses ofthe polypeptide.

As used herein, the term “intact antibody” means an antibody that has atleast two heavy (H) chain polypeptides and two light (L) chainpolypeptides interconnected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor V_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH₁, CH₂ and CH₃. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxyl-terminus in the following order: FR₁, CDR₁,FR₂, CDR₂, FR₃, CDR₃, FR₄. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

As used herein, the term “immune response” refers to the concertedaction of lymphocytes, antigen presenting cells, phagocytic cells,granulocytes, and soluble macromolecules produced by the above cells orthe liver (including antibodies, cytokines, and complement) that resultsin selective damage to, destruction of, or elimination from the humanbody of cancerous cells, metastatic tumor cells, malignant melanoma,invading pathogens, cells or tissues infected with pathogens, or, incases of autoimmunity or pathological inflammation, normal human cellsor tissues.

As used herein, the terms “immunologically cross-reactive” and“immunologically-reactive” are used interchangeably to mean an antigenwhich is specifically reactive with an antibody which was generatedusing the same (“immunologically-reactive”) or different(“immunologically cross-reactive”) antigen. Generally, the antigen isgelsolin polypeptide, a variant or subsequence thereof.

As used herein, the term “immunologically-reactive conditions” meansconditions which allow an antibody, generated to a particular epitope ofan antigen, to bind to that epitope to a detectably greater degree thanthe antibody binds to substantially all other epitopes, generally atleast two times above background binding, preferably at least five timesabove background. Immunologically-reactive conditions are dependent uponthe format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See, Harlow & Lane, Antibodies, ALaboratory Manual (Cold Spring Harbor Publications, New York, 1988) fora description of immunoassay formats and conditions.

As used herein, the term “lymphocyte” means any of the mononuclear,nonphagocytic leukocytes, found in the blood, lymph, and lymphoidtissues, e.g., B and T lymphocytes.

As used herein, the term “medical condition” includes, but is notlimited to, any condition or disease manifested as one or more physicaland/or psychological symptoms for which treatment and/or prevention isdesirable, and includes previously and newly identified diseases andother disorders. For example, a medical condition may be hepatitis, SLE,cancer, septic shock, stroke, heart infarction, and side effects ofchemotherapy and radiation therapy.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. For example, a monoclonal antibody can be an antibodythat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.A monoclonal antibody composition displays a single binding specificityand affinity for a particular epitope. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. Furthermore,in contrast to conventional (polyclonal) antibody preparations whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. The modifier “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method.Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including, e.g., but not limited to, hybridoma,recombinant, and phage display technologies. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

As used herein, the term “pharmaceutically-acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal compounds, isotonic and absorption delayingcompounds, and the like, compatible with pharmaceutical administration.

As used herein, the term “polyclonal antibody” means a preparation ofantibodies derived from at least two (2) different antibody-producingcell lines. The use of this term includes preparations of at least two(2) antibodies that contain antibodies that specifically bind todifferent epitopes or regions of an antigen.

As used herein, the term “polynucleotide” or “nucleic acid” means anyRNA or DNA, which may be unmodified or modified RNA or DNA.Polynucleotides include, without limitation, single- and double-strandedDNA, DNA that is a mixture of single- and double-stranded regions,single- and double-stranded RNA, RNA that is mixture of single- anddouble-stranded regions, and hybrid molecules comprising DNA and RNAthat may be single-stranded or, more typically, double-stranded or amixture of single- and double-stranded regions. In addition,polynucleotide refers to triple-stranded regions comprising RNA or DNAor both RNA and DNA. The term polynucleotide also includes DNAs or RNAscontaining one or more modified bases and DNAs or RNAs with backbonesmodified for stability or for other reasons. In a particular embodiment,the polynucleotide contains polynucleotide sequences from a gelsolingene.

As used herein, the terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. Polypeptide refers to both short chains,commonly referred to as peptides, glycopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.Polypeptides include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature. In aparticular embodiment, the polypeptide contains polypeptide sequencesfrom a gelsolin protein.

As used herein, the term “population” may be any group of at least twoindividuals. A population may include, e.g. but is not limited to, areference population, a population group, a family population, aclinical population, and a same sex population.

As used herein, the term “recombinant” when used with reference, e.g.,to a cell, or nucleic acid, protein, or vector, indicates that the cell,nucleic acid, protein or vector, has been modified by the introductionof a heterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the material is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all.

As used herein, the term “reference standard” is the pattern ofexpression of one or more genes or proteins observed in either areference standard population or a single subject prior toadministration of a compound.

As used herein, the phrase “salvage receptor binding epitope” refers toan epitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃,or IgG₄) that is responsible for increasing the in vivo serum half-lifeof the IgG molecule To increase the serum half life of the antibody, onemay incorporate a salvage receptor binding epitope into the antibody(especially an antibody fragment) as described in U.S. Pat. No.5,739,277, for example.

As used herein, the terms “single chain antibodies” or “single chain Fv(scFv)” refer to an antibody fusion molecule of the two domains of theFv fragment, V_(L) and V_(H). Although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv). See, e.g., Bird et al., Science 242: 423-426, 1988; and Hustonet al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883, 1988). Such singlechain antibodies are included by reference to the term “antibody”fragments, and can be prepared by recombinant techniques or enzymatic orchemical cleavage of intact antibodies.

As used herein, the term “small molecule” means a composition that has amolecular weight of less than about 5 kDa and more preferably less thanabout 2 kDa. Small molecules can be, e.g., nucleic acids, peptides,polypeptides, glycopeptides, peptidomimetics, carbohydrates, lipids,lipopolysaccharides, combinations of these, or other organic orinorganic molecules.

As used herein, the term “specific binding” means the contact between agelsolin binding agent and an antigen with a binding affinity of atleast 10⁻⁶ M. Preferred binding agents bind with affinities of at leastabout 10⁻⁷ M, and preferably 10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or10⁻¹² M.

As used herein, the term “subject” means that preferably the subject isa mammal, such as a human, but can also be an animal, e.g., domesticanimals (e.g., dogs, cats and the like), farm animals (e.g., cows,sheep, pigs, horses and the like) and laboratory animals (e.g., monkey,rats, mice, rabbits, guinea pigs and the like).

As used herein, the term “substitution” is one of mutations that isgenerally used in the art. Those substitution variants have at least oneamino acid residue in the gelsolin binding antibody molecule replaced bya different residue. The sites of greatest interest for substitutionalmutagenesis include the hypervariable regions, but FR alterations arealso contemplated. “Conservative substitutions” are shown in the Tablebelow under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 2,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

As used herein, the term “reference standard population” means apopulation characterized by one or more biological characteristics,e.g., drug responsiveness, genotype, haplotype, phenotype, etc.

As used herein, a “test sample” means a biological sample obtained froma subject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell or tissue sample, sample from culture orgrowth media, or isolated nucleic acid or polypeptide derived therefrom.

TABLE 2 Amino Acid Substitutions Original Residue ExemplarySubstitutions Preferred Substitutions Ala (A) val; leu; ile val Arg (R)lys; gln; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asnglu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly(G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;phe; leu norleucine Leu (L) norleucine; ile; val; met; ile ala; phe Lys(K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile;ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W)tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe;ala; leu norleucine

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Specifically, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibody variantsthus generated are displayed in a monovalent fashion from filamentousphage particles as fusions to the gene III product of M13 packagedwithin each particle. The phage-displayed variants are then screened fortheir biological activity (e.g. binding affinity) as herein disclosed.In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding gelsolin. Alternatively, or additionally, it may be beneficialto analyze a crystal structure of the antigen-antibody complex toidentify contact points between the antibody and gelsolin. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with similar or superior properties in one or morerelevant assays may be selected for further development. The inventionincludes antibody variants with one or more amino acid substitution(s),especially conservative substitutions, to the hypervariable domains ofthe immunoglobulin heavy or light chain defined by hybridomas GC1C10,GN3E9, GF2D6, having CGMCC Accession Numbers 2114, 2115, and 2116,respectively, provided that the antibody variant possesses the desiredproperties.

As used herein, the term “therapeutic agent” is intended to mean acompound that, when present in an effective amount, produces a desiredtherapeutic effect on a subject in need thereof.

As used herein, the terms “treating” or “treatment” or “alleviation”refers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. A subject is successfully“treated” for a disorder characterized by decreased gelsolin levels if,after receiving a therapeutic amount of native or recombinant gelsolinaccording to the methods of the present invention, the subject showsobservable and/or measurable reduction in or absence of one or moresigns and symptoms of a particular disease or condition. For example,for cancer, reduction in the number of cancer cells or absence of thecancer cells; reduction in the tumor size; inhibition (i.e., slow tosome extent and preferably stop) of tumor metastasis; inhibition, tosome extent, of tumor growth; increase in length of remission, and/orrelief to some extent, one or more of the symptoms associated with thespecific cancer; reduced morbidity and mortality, and improvement inquality of life issues.

As used herein, the term “variable” refers to the fact that certainsegments of the variable domains differ extensively in sequence amongantibodies. The V domain mediates antigen binding and define specificityof a particular antibody for its particular antigen. However, thevariability is not evenly distributed across the amino acid span of thevariable domains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting β-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

Compositions of the Invention

Gelsolin Binding Agents. In one aspect, the present invention providesgelsolin binding agent compositions, a.k.a., the binding agent. In oneembodiment, the binding agent of the invention is an intact antibodydirected to a gelsolin polypeptide, homolog, fragment, or derivativethereof. The binding agents of interest may be ones which bindspecifically to free, full-length, active gelsolin, but do not“substantively” (or “substantially”) bind gelsolin which are bound toactin. In such embodiments, the extent of binding of the binding agentof the invention to these proteins will be less than about 10%,preferably, or less than about 5%, or less than about 1%, as determinedby fluorescence activated cell sorting (FACS) analysis, ELISA, Westernblot, or radioimmunoassay.

Prior efforts to generate monoclonal antibodies with defined epitopes(in particular, eptiopes associated with functional gelsolin) have beenlargely unsuccessful. Gelsolin is a highly conserved protein and highlyhomologous among species. Gelsolin is also abundant in plasma, whichrequires that it be well-tolerated by the immune system. Furthermore,due to the fact that gelsolin is a major actin binding protein, theexposed epitopes are limited by the complexing of gelsolin with actinand other plasma proteins. Although some monoclonal antibodies togelsolin have been produced (see Hiyoshi et al., Biochem Mol Biol Int.32:755-62 (1994)), there is no immunoassay for quantitative measurementof plasma gelsolin.

The present inventors have discovered a strategy to design gelsolinbinding agents using various forms of human gelsolin proteins for bothimmunization and screening, including native gelsolin, recombinantfull-length gelsolin, and N- and C-terminal gelsolin fragments.Moreover, the inventors' strategy is designed to break the immunetolerance to common epitopes of human gelsolin using modulators of theimmune response. The result of this strategy are gelsolin binding agentswith defined epitopes that allow for rapid, accurate, and quantitativeassays for plasma gelsolin which can be used in a clinical setting.

Binding agents of the present invention can be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the bindingagent, e.g., a region of the gelsolin polypeptide that is located on thesurface of the polypeptide (e.g., a hydrophilic region). In oneembodiment, the invention provides gelsolin binding agents, e.g.,antibodies or antibody-related polypeptides directed to a gelsolinpolypeptide (a.k.a., a target polypeptide) comprising one or more aminoacid sequences selected from the group consisting of: FAQGALKSED (SEQ IDNO.: 2), SEPDGFWEAL (SEQ ID NO.: 3), ACSNKIGRFV (SEQ ID NO.: 4).

In select embodiments, the invention provides the gelsolin bindingagents summarized in Table 3.

TABLE 3 Select Gelsolin Receptor-Binding Agents Binding Agent TypeDescription GN3E9 Murine Monoclonal Murine monoclonal antibody directedAntibody to an epitope comprising a polypeptidesequence of FAQGALKSED (SEQ ID NO.: 2). GC1C10 Murine MonoclonalMurine monoclonal antibody directed Antibodyto an epitope comprising a polypeptidesequence of SEPDGFWEAL (SEQ ID NO.: 3). GF2D6 Murine MonoclonalMurine monoclonal antibody directed Antibodyto a epitope with a polypeptide sequence of ACSNKIGRFV (SEQ ID NO.: 4).

Deposits of biological materials associated with the gelsolin bindingagents summarized in Table 5 (above) were made with the China GeneralMicrobiological Culture Collection Center (CGMCC), China Committee forCulture Collection of Microorganisms, P.O. Box 2714, Beijing 100080, ThePeople's Republic of China as detailed in Table 4 below.

TABLE 4 Biological Deposits Accession Name of Deposit Materials DateNumber GN3E9 Mouse-mouse hybridoma Jul. 20, 2007 2115 GC1C10 Mouse-mousehybridoma Jul. 20, 2007 2114 GF2D6 Mouse-mouse hybridoma Jul. 20, 20072116

In another embodiment, the present invention affords a method ofelucidating other epitopes of gelsolin, which can be used for generationof an antibody having the desired characteristics of binding to activegelsolin, but not gelsolin bound to actin. The binding agents directedagainst the epitope may have a differing variable or CDR region butshould have the binding and functional characteristics of the antibodiesof the present invention. As a means for targeting antibody production,hydropathy plots showing regions of hydrophilicity and hydrophobicitycan be generated by any method well known in the art, including, e.g.,the Kyte Doolittle or the Hopp Woods methods, either with or withoutFourier transformation (see, e.g., Hopp and Woods, Proc. Nat. Acad. Sci.USA 78: 3824-3828 (1981); Kyte and Doolittle, J. Mol. Biol. 157: 105-142(1982)). The epitope(s) or polypeptide portion(s) can be specified asdescribed herein, e.g., by N-terminal and C-terminal positions, by sizein contiguous amino acid residues. The present invention includesbinding agents that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same. The presentinvention includes binding agents that specifically bind epitopes whichare conformational epitopes or nonconformational epitopes. As notedabove, conformational epitopes or nonconformational epitopes aredistinguished in that the binding to the former but not the latter islost in the presence of denaturing solvents.

Binding agents of the present invention can also be described orspecified in terms of their cross-reactivity. Binding agents that do notbind any other analog, ortholog, or homolog of the target polypeptide ofthe present invention are included. Binding agents that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. Further includedin the present invention are binding agents which only bind polypeptidesencoded by polynucleotides which hybridize to a polynucleotide of thepresent invention under stringent hybridization conditions (as describedherein).

Binding agents of the present invention can also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or K_(d) less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,10⁻¹⁴, 5×10⁻¹⁵M, and 10⁻¹⁵M. In one embodiment, the invention providesgelsolin binding agents that at least bind human gelsolin with a K_(d)value of no higher than 1×10⁻⁸, preferably a K_(d) value no higher thanabout 1×10⁻⁹.

Gelsolin binding agents within the scope of the present inventioninclude, e.g., but are not limited to, monoclonal, polyclonal, chimeric,humanized, diabody, and human monoclonal and human polyclonal antibodieswhich specifically bind the target polypeptide, a homolog, derivative ora fragment thereof. As used herein, a “gelsolin-like polypeptide” meansa polypeptide that is different from gelsolin polypeptide but which isimmunologically-reactive with a gelsolin binding agent of the invention.A gelsolin-like polypeptide may be derived from the same organism or adifferent organism as a gelsolin polypeptide. A gelsolin-likepolypeptide may be encoded by the same gene or a different gene as agelsolin polypeptide. The antibodies useful as binding agents of thepresent invention include, e.g., but are not limited to, IgG (includingIgG₁, IgG₂, IgG₃, and IgG₄), IgA (including IgA₁ and IgA₂), IgD, IgE, orIgM, and IgY.

In another embodiment, the binding agent of the invention is anantibody-related polypeptide directed to gelsolin polypeptide, homologor derivative thereof. Typically, the antigen-binding region of abinding agent, e.g., the anti-gelsolin binding region, will be mostcritical in specificity and affinity of binding of the binding agent ofthe invention. In some embodiments, the gelsolin binding agent is ananti-gelsolin polypeptide antibody, such as an anti-gelsolin polypeptidemonoclonal antibody, an anti-gelsolin polypeptide chimeric antibody, andan anti-gelsolin polypeptide humanized antibody which have been modifiedby, e.g., deleting, adding, or substituting portions of the antibody.For example, an anti-gelsolin polypeptide antibody intended meant toincrease half-life, e.g., serum half-life, stability or affinity of theantibody.

In one embodiment, selection of antibodies that are specific to aparticular domain of a gelsolin polypeptide is facilitated by generationof hybridomas that bind to the fragment of a gelsolin polypeptidepossessing such a domain. Thus, gelsolin binding agents which areantibodies that are specific for a desired domain within a gelsolinpolypeptide, or derivatives, fragments, analogs or homologs thereof, arealso provided herein.

The present invention further includes antibodies which areanti-idiotypic to the binding agents of the present invention. Thebinding agents of the present invention can be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific binding agentscan be specific for different epitopes of a gelsolin polypeptide of thepresent invention or can be specific for both a gelsolin polypeptide ofthe present invention as well as for heterologous compositions, such asa heterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J.Immunol. 147: 60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893,5,601,819, 4,714,681, 4,925,648; 6,106,835; Kostelny et al., J. Immunol.148: 1547-1553 (1992). The binding agents of the invention can be fromany animal origin including birds and mammals. Preferably, the bindingagents are human, murine, rabbit, goat, guinea pig, camel, horse, orchicken.

The binding agents of the present invention can be used either alone orin combination with other compositions. For example, the gelsolinbinding agents of the invention can be used in combination with one ormore anti-gelsolin antibodies known in the art, e.g., but not limited toantibody GS-2C4 (Sigma-Aldrich, Cat. No. G4896; Afify and Werness. Appl.Immunohistochem. 6:30, (1998)).

The gelsolin binding agents of the present invention can further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, gelsolin binding agents of the present invention can berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

In certain embodiments, the gelsolin binding agents of the presentinvention are anti-gelsolin antibodies or anti-gelsolin antibody-relatedpolypeptides that are coupled or conjugated to one or more therapeuticor cytotoxic moieties to yield a gelsolin binding agent conjugateprotein of the invention. The gelsolin binding agent conjugate proteinof the invention can be used to modify a given biological response orcreate a biological response (e.g., to recruit effector cells). Thetherapeutic moiety is not to be construed as limited to classicalchemical therapeutic agents. For example, the therapeutic moiety can bea protein or polypeptide possessing a desired biological activity. Suchproteins can include, e.g., an enzymatically active toxin, or activefragment thereof, such as abrin, ricin A, Pseudomonas exotoxin, ordiphtheria toxin; a protein such as tumor necrosis factor orinterferon-alpha; or, biological response modifiers such as, e.g.,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Methods of Preparing a Gelsolin-Binding Agents of the Invention

General Overview. Initially, a target polypeptide is chosen to which abinding agent of the invention (e.g., anti-gelsolin receptor antibody)can be raised. Techniques for generating binding agents directed totarget polypeptides are well known to those skilled in the art. Examplesof such techniques include, e.g., but are not limited to, thoseinvolving display libraries, xeno or humab mice, hybridomas, and thelike. Target polypeptides within the scope of the present inventioninclude any polypeptide or polypeptide derivative which is capable ofexhibiting antigenicity. Examples include, but are not limited togelsolin, peptides, polypeptides, and fragments thereof.

It should be understood that not only are naturally-occurring antibodiessuitable as binding agents for use in accordance with the presentdisclosure, but recombinantly engineered antibodies and antibodyfragments, e.g., antibody-related polypeptides, which are directed togelsolin polypeptide and fragments thereof are also suitable.

Binding agents, e.g., anti-gelsolin antibodies, that can be subjected tothe techniques set forth herein include monoclonal and polyclonalantibodies, and antibody fragments such as Fab, Fab′, F(ab′)₂, Fd, scFv,diabodies, antibody light chains, antibody heavy chains and/or antibodyfragments. Methods useful for the high yield production of antibodyFv-containing polypeptides, e.g., Fab′ and F(ab′)₂ antibody fragmentshave been described. See U.S. Pat. No. 5,648,237.

Generally, a binding agent is obtained from an originating species. Moreparticularly, the nucleic acid or amino acid sequence of the variableportion of the light chain, heavy chain or both, of an originatingspecies antibody having specificity for a target polypeptide antigen isobtained. Originating species is any species which was useful togenerate the binding agent of the invention or library of bindingagents, e.g., rat, mice, rabbit, chicken, monkey, human, and the like.

In preferred embodiments, gelsolin binding agents are anti-gelsolinantibodies. Phage or phagemid display technologies are useful techniquesto derive the binding agents of the present invention. Anti-gelsolinantibodies useful in the present invention are “human antibodies,”(e.g., antibodies isolated from a human) or “human sequence antibodies.”Human antibodies can be made by a variety of methods known in the artincluding phage display methods. See also, U.S. Pat. Nos. 4,444,887,4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO 98/50433, WO98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.Methods useful for the identification of nucleic acid sequences encodingmembers of multimeric polypeptide complex by screening polyphageparticles have been described. Rudert et al., U.S. Pat. No. 6,667,150.Also, recombinant immunoglobulins can be produced. Cabilly, U.S. Pat.No. 4,816,567; Cabilly et al., U.S. Pat. No. 6,331,415 and Queen et al.,Proc. Natl. Acad. Sci. USA 86: 10029-10033, 1989. Techniques forgenerating and cloning monoclonal antibodies are well known to thoseskilled in the art. The gelsolin binding agents of the inventionpreferably have a high immunoreactivity, that is, percentages ofantibodies molecules that are correctly folded so that they canspecifically bind their target antigen. Expression of sequences encodingbinding agents, e.g., antibodies of the invention, can be carried out inE. coli as described below. Such expression usually results inimmunoreactivity of at least 80%, 90%, 95% or 99%.

Certain truncations of these proteins or genes perform the regulatory orenzymatic functions of the full sequence protein or gene. For example,the nucleic acid sequences coding therefore can be altered bysubstitutions, additions, deletions or multimeric expression thatprovide for functionally equivalent proteins or genes. Due to thedegeneracy of nucleic acid coding sequences, other sequences whichencode substantially the same amino acid sequences as those of thenaturally occurring proteins may be used in the practice of the presentinvention. These include, but are not limited to, nucleic acid sequencesincluding all or portions of the nucleic acid sequences encoding theabove polypeptides, which are altered by the substitution of differentcodons that encode a functionally equivalent amino acid residue withinthe sequence, thus producing a silent change. It is appreciated that thenucleotide sequence of an immunoglobulin according to the presentinvention tolerates sequence homology variations of up to 25% ascalculated by standard methods (“Current Methods in Sequence Comparisonand Analysis,” Macromolecule Sequencing and Synthesis, Selected Methodsand Applications, pp. 127-149, 1998, Alan R. Liss, Inc.) so long as sucha variant forms an operative antibody which recognizes gelsolin orgelsolin-like polypeptides. For example, one or more amino acid residueswithin a polypeptide sequence can be substituted by another amino acidof a similar polarity which acts as a functional equivalent, resultingin a silent alteration. Substitutes for an amino acid within thesequence may be selected from other members of the class to which theamino acid belongs. For example, the nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan and methionine. The polar neutral amino acids includeglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Also included within the scopeof the present invention are proteins or fragments or derivativesthereof which are differentially modified during or after translation,e.g., by glycosolation, protolytic cleavage, linkage to an antibodymolecule or other cellular ligands, etc. Additionally, an inhibitorencoding nucleic acid sequence can be mutated in vitro or in vivo tocreate and/or destroy translation, initiation, and/or terminationsequences or to create variations in coding regions and/or form newrestriction endonuclease sites or destroy pre-existing ones, tofacilitate further in vitro modification. Any technique for mutagenesisknown in the art can be used, including but not limited to in vitro sitedirected mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers(Pharmacia), and the like.

Preparation of Polyclonal Antisera and Immunogens. Methods of generatingantibodies or antibody fragments of the invention typically includeimmunizing a subject (generally a non-human subject such as a mouse orrabbit) with a purified gelsolin or gelsolin-like polypeptide or homologor fragment thereof or with a cell expressing the gelsolin orgelsolin-like polypeptide or homolog or fragment thereof. Anyimmunogenic portion of the gelsolin polypeptide can be employed as theimmunogen. An appropriate immunogenic preparation can contain, e.g., arecombinantly-expressed gelsolin polypeptide or a chemically-synthesizedgelsolin polypeptide. An isolated gelsolin polypeptide, or a portion orfragment thereof, can be used as an immunogen to generate a gelsolinbinding agent that binds to the gelsolin polypeptide, or a portion orfragment using standard techniques for polyclonal and monoclonalantibody preparation. The full-length gelsolin polypeptide can be usedor, alternatively, the invention provides for the use of the gelsolinpolypeptide fragments as immunogens. The gelsolin polypeptide comprisesat least four amino acid residues of the amino acid sequence shown inSEQ ID NO.: 1, and encompasses an epitope of the gelsolin polypeptidesuch that an antibody raised against the peptide forms a specific immunecomplex with the gelsolin polypeptide. Preferably, the antigenic peptidecomprises at least 5, 8, 10, 15, 20, or 30 amino acid residues. Longerantigenic peptides are sometimes preferable over shorter antigenicpeptides, depending on use and according to methods well known to thoseskilled in the art. Typically, the immunogen will be at least about 8amino acyl residues in length, and preferably at least about 10 acylresidues in length. Multimers of a given epitope are sometimes moreeffective than a monomer.

If needed, the immunogenicity of the gelsolin polypeptide (or fragmentthereof) can be increased by fusion or conjugation to a hapten such askeyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptensare known in the art. One can also combine the gelsolin polypeptide witha conventional adjuvant such as Freund's complete or incomplete adjuvantto increase the subject's immune reaction to the polypeptide. Variousadjuvants used to increase the immunological response include, but arenot limited to, Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), human adjuvants such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory compounds. Thesetechniques are standard in the art.

For convenience, immune responses are often described in the presentinvention as being either “primary” or “secondary” immune responses. Aprimary immune response, which is also described as a “protective”immune response, refers to an immune response produced in an individualas a result of some initial exposure (e.g., the initial “immunization”)to a particular antigen, e.g., a gelsolin polypeptide. Such animmunization can occur, e.g., as the result of some natural exposure tothe antigen (e.g., from initial infection by some pathogen that exhibitsor presents the antigen) or from antigen presented by cancer cells ofsome tumor in the individual (e.g., malignant melanoma). Alternatively,the immunization can occur as a result of vaccinating the individualwith a vaccine containing the antigen. For example, the vaccine can be agelsolin vaccine comprising one or more antigens from a gelsolinpolypeptide or a gelsolin-like polypeptide.

A primary immune response can become weakened or attenuated over timeand can even disappear or at least become so attenuated that it cannotbe detected. Accordingly, the present invention also relates to a“secondary” immune response, which is also described here as a “memoryimmune response.” The term secondary immune response refers to an immuneresponse elicited in an individual after a primary immune response hasalready been produced.

Thus, a secondary or immune response can be elicited, e.g., to enhancean existing immune response that has become weakened or attenuated, orto recreate a previous immune response that has either disappeared orcan no longer be detected. The secondary or memory immune response canbe either a humoral (antibody) response or a cellular response. Asecondary or memory humoral response occurs upon stimulation of memory Bcells that were generated at the first presentation of the antigen.Delayed type hypersensitivity (DTH) reactions are a type of cellularsecondary or memory immune response that are mediated by CD4⁺ cells. Afirst exposure to an antigen primes the immune system and additionalexposure(s) results in a DTH.

Following appropriate immunization, the gelsolin binding agent, e.g.,anti-gelsolin polyclonal antibody can be prepared from the subject'sserum. If desired, the antibody molecules directed against the gelsolinpolypeptide can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as polypeptide Achromatography to obtain the IgG fraction.

Monoclonal Antibody. In one embodiment of the present invention, thebinding agent is an anti-gelsolin monoclonal antibody. For example, insome embodiments, the anti-gelsolin monoclonal antibody may be a humanor a mouse anti-gelsolin monoclonal antibody. For preparation ofmonoclonal antibodies directed towards a particular gelsolinpolypeptide, or derivatives, fragments, analogs or homologs thereof, anytechnique that provides for the production of antibody molecules bycontinuous cell line culture can be utilized. Such techniques include,but are not limited to, the hybridoma technique (see, e.g., Kohler &Milstein, 1975. Nature 256: 495-497); the trioma technique; the humanB-cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol.Today 4: 72) and the EBV hybridoma technique to produce human monoclonalantibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES ANDCANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies can be utilized in the practice of the invention and can beproduced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc.Natl. Acad. Sci. USA 80: 2026-2030) or by transforming human B-cellswith Epstein Barr Virus in vitro (see, e.g., Cole, et al., 1985. In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). For example, a population of nucleic acids that encode regionsof antibodies can be isolated. PCR utilizing primers derived fromsequences encoding conserved regions of antibodies is used to amplifysequences encoding portions of antibodies from the population and thenreconstruct DNAs encoding antibodies or fragments thereof, such asvariable domains, from the amplified sequences. Such amplified sequencesalso can be fused to DNAs encoding other proteins—e.g., a bacteriophagecoat, or a bacterial cell surface protein—for expression and display ofthe fusion polypeptides on phage or bacteria. Amplified sequences canthen be expressed and further selected or isolated based, e.g., on theaffinity of the expressed antibody or fragment thereof for an antigen orepitope present on the gelsolin polypeptide. Alternatively, hybridomasexpressing anti-gelsolin monoclonal antibodies can be prepared byimmunizing a subject and then isolating hybridomas from the subject'sspleen using routine methods. See, e.g., Milstein et al., (Galfre andMilstein, Methods Enzymol (1981) 73: 3-46). Screening the hybridomasusing standard methods will produce monoclonal antibodies of varyingspecificity (i.e., for different epitopes) and affinity. A selectedmonoclonal antibody with the desired properties, e.g., gelsolin binding,can be used as expressed by the hybridoma, it can be bound to a moleculesuch as polyethylene glycol (PEG) to alter its properties, or a cDNAencoding it can be isolated, sequenced and manipulated in various ways.Synthetic dendroineric trees can be added a reactive amino acid sidechains, e.g., lysine to enhance the immunogenic properties of thegelsolin polypeptide. Also, CPG-dinucleotide technique can be used toenhance the immunogenic properties of the gelsolin polypeptide. Othermanipulations include substituting or deleting particular amino acylresidues that contribute to instability of the antibody during storageor after administration to a subject, and affinity maturation techniquesto improve affinity of the antibody of the gelsolin polypeptide.

Hybridoma Technique. In one embodiment, the binding agent of theinvention is an anti-gelsolin monoclonal antibody produced by ahybridoma which includes a B cell obtained from a transgenic non-humananimal, e.g., a transgenic mouse, having a genome comprising a humanheavy chain transgene and a light chain transgene fused to animmortalized cell. Hybridoma techniques include those known in the artand taught in Harlow et al., Antibodies: A Laboratory Manual Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 349 (1988); Hammerling etal., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Othermethods for producing hybridomas and monoclonal antibodies are wellknown to those of skill in the art.

Phage Display Technique. As noted above, the binding agents of thepresent invention can be produced through the application of recombinantDNA and phage display technology. For example, binding agents of theinvention, e.g., anti-gelsolin antibodies, can be prepared using variousphage display methods known in the art. In phage display methods,functional antibody domains are displayed on the surface of a phageparticle which carries polynucleotide sequences encoding them. Phagewith a desired binding property are selected from a repertoire orcombinatorial antibody library (e.g., human or murine) by selectingdirectly with antigen, typically antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 with Fab, Fv or disulfide stabilized Fvantibody domains are recombinantly fused to either the phage gene III orgene VIII protein. In addition, methods can be adapted for theconstruction of Fab expression libraries (see, e.g., Huse, et al.,Science 246: 1275-1281, 1989) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor a gelsolin polypeptide, e.g., a polypeptide or derivatives,fragments, analogs or homologs thereof. Other examples of phage displaymethods that can be used to make the binding agents of the presentinvention include those disclosed in Huston et al., Proc. Natl. Acad.Sci. U.S.A., 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad.Sci. U.S.A., 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995;Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al.,Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280,1994; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO92/18619; WO 93/11236; WO 95/15982; WO 95/20401; WO 96/06213; WO92/01047 (Medical Research Council et al.); WO 97/08320 (Morphosys); WO92/01047 (CAT/MRC); WO 91/17271 (Affymax); and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743.Methods useful for displaying polypeptides on the surface ofbacteriophage particles by attaching the polypeptides via disulfidebonds have been described by Lohning, U.S. Pat. No. 6,753,136. Asdescribed in the above references, after phage selection, the antibodycoding regions from the phage can be isolated and used to generate wholeantibodies, including human antibodies, or any other desired antigenbinding fragment, and expressed in any desired host including mammaliancells, insect cells, plant cells, yeast, and bacteria. For example,techniques to recombinantly produce Fab, Fab′ and F(ab′)₂ fragments canalso be employed using methods known in the art such as those disclosedin WO 92/22324; Mullinax et al., BioTechniques 12: 864-869, 1992; andSawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240:1041-1043, 1988.

Generally, hybrid antibodies or hybrid antibody fragments that arecloned into a display vector can be selected against the appropriateantigen in order to identify variants that maintained good bindingactivity, because the antibody or antibody fragment will be present onthe surface of the phage or phagemid particle. See e.g. Barbas III etal., Phage Display, A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001). However, other vector formatscould be used for this process, such as cloning the antibody fragmentlibrary into a lytic phage vector (modified T7 or Lambda Zap systems)for selection and/or screening.

Expression of Recombinant Gelsolin-Binding Agent. As noted above, thebinding agents of the present invention can be produced through theapplication of recombinant DNA technology. Recombinant polynucleotideconstructs encoding a gelsolin binding agent of the present inventiontypically include an expression control sequence operably-linked to thecoding sequences of anti-gelsolin antibody chains, includingnaturally-associated or heterologous promoter regions. As such, anotheraspect of the invention includes vectors containing one or more nucleicacid sequences encoding a gelsolin binding agent of the presentinvention. For recombinant expression of one or more the polypeptides ofthe invention, the nucleic acid containing all or a portion of thenucleotide sequence encoding the gelsolin binding agent is inserted intoan appropriate cloning vector, or an expression vector (i.e., a vectorthat contains the necessary elements for the transcription andtranslation of the inserted polypeptide coding sequence) by recombinantDNA techniques well known in the art and as detailed below. Methods forproducing diverse populations of vectors have been described by Lerneret al., U.S. Pat. Nos. 6,291,160; 6,680,192.

In general, expression vectors useful in recombinant DNA techniques areoften in the form of plasmids. In the present specification, “plasmid”and “vector” can be used interchangeably as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors that are not technicallyplasmids, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions. Such viral vectors permit infection of a subjectand expression in that subject of a compound. Preferably, the expressioncontrol sequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences encoding the gelsolin binding agent, and the collection andpurification of the gelsolin binding agent, e.g., cross-reactinganti-gelsolin antibodies. See, generally, U.S. Application No.20020199213. These expression vectors are typically replicable in thehost organisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markers,e.g., ampicillin-resistance or hygromycin-resistance, to permitdetection of those cells transformed with the desired DNA sequences.Vectors can also encode signal peptide, e.g., pectate lyase, useful todirect the secretion of extracellular antibody fragments. See U.S. Pat.No. 5,576,195.

The recombinant expression vectors of the invention comprise a nucleicacid encoding a compound with gelsolin binding properties in a formsuitable for expression of the nucleic acid in a host cell, which meansthat the recombinant expression vectors include one or more regulatorysequences, selected on the basis of the host cells to be used forexpression that is operatively-linked to the nucleic acid sequence to beexpressed. Within a recombinant expression vector, “operably-linked” isintended to mean that the nucleotide sequence of interest is linked tothe regulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, e.g., in Goeddel,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcell and those that direct expression of the nucleotide sequence only incertain host cells (e.g., tissue-specific regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of polypeptide desired,etc. Typical regulatory sequences useful as promoters of recombinantpolypeptide expression (e.g., gelsolin binding agents), include, e.g.,but are not limited to, 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization. In one embodiment, a polynucleotideencoding a gelsolin binding agent of the invention is operably-linked toan ara B promoter and expressible in a host cell. See U.S. Pat. No.5,028,530. The expression vectors of the invention can be introducedinto host cells to thereby produce polypeptides or peptides, includingfusion polypeptides, encoded by nucleic acids as described herein (e.g.,gelsolin binding agents, etc.).

Another aspect of the invention pertains to gelsolin bindingagent-expressing host cells, which contain a nucleic acid encoding oneor more gelsolin binding agents. The recombinant expression vectors ofthe invention can be designed for expression of a gelsolin binding agentin prokaryotic or eukaryotic cells. For example, a gelsolin bindingagent can be expressed in bacterial cells such as Escherichia coli,insect cells (using baculovirus expression vectors), fungal cells, e.g.,yeast, yeast cells or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, e.g. using T7 promoter regulatory sequences and T7 polymerase.Methods useful for the preparation screening of polypeptides havingpredetermined property, e.g., gelsolin binding agents, via expression ofstochastically generated polynucleotide sequences has been described.See U.S. Pat. Nos. 5,763,192; 5,723,323; 5,814,476; 5,817,483;5,824,514; 5,976,862; 6,492,107; 6,569,641.

Expression of polypeptides in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion polypeptides.Fusion vectors add a number of amino acids to a polypeptide encodedtherein, usually to the amino terminus of the recombinant polypeptide.Such fusion vectors typically serve three purposes: (i) to increaseexpression of recombinant polypeptide; (ii) to increase the solubilityof the recombinant polypeptide; and (iii) to aid in the purification ofthe recombinant polypeptide by acting as a ligand in affinitypurification. Often, in fusion expression vectors, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant polypeptide to enable separation of the recombinantpolypeptide from the fusion moiety subsequent to purification of thefusion polypeptide. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione5-transferase (GST), maltose E binding polypeptide, or polypeptide A,respectively, to the target recombinant polypeptide.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89). Methods for targetedassembly of distinct active peptide or protein domains to yieldmultifunctional polypeptides via polypeptide fusion has been describedby Pack et al., U.S. Pat. Nos. 6,294,353; 6,692,935. One strategy tomaximize recombinant polypeptide expression, e.g., a gelsolin bindingagent, in E. coli is to express the polypeptide in host bacteria with animpaired capacity to proteolytically cleave the recombinant polypeptide.See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategyis to alter the nucleic acid sequence of the nucleic acid to be insertedinto an expression vector so that the individual codons for each aminoacid are those preferentially utilized in the expression host, e.g., E.coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118).Such alteration of nucleic acid sequences of the invention can becarried out by standard DNA synthesis techniques.

In another embodiment, the gelsolin binding agent expression vector is ayeast expression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982),pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). Alternatively, a gelsolin binding agent can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of polypeptides, e.g., gelsolin binding agents,in cultured insect cells (e.g., SF9 cells) include the pAc series(Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series(Lucklow and Summers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid encoding a gelsolin bindingagent of the invention is expressed in mammalian cells using a mammalianexpression vector. Examples of mammalian expression vectors include,e.g., but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) andpMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, adenovirus 2, cytomegalovirus, andsimian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells useful for expression of the gelsolinbinding agents of the present invention. See, e.g., Chapters 16 and 17of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,Genes Dev. 1: 268-277, 1987), lymphoid-specific promoters (Calame andEaton, Adv. Immunol. 43: 235-275, 1988), in particular promoters of Tcell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, Cell 33: 741-748, 1983.), neuron-specific promoters (e.g.,the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA86: 5473-5477, 1989), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, Science249: 374-379, 1990) and the α-fetoprotein promoter (Campes and Tilghman,Genes Dev. 3: 537-546, 1989).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, agelsolin binding agent can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells. Mammalian cells are apreferred host for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes ToClones, (VCH Publishers, NY, 1987). A number of suitable host cell linescapable of secreting intact heterologous proteins have been developed inthe art, and include Chinese hamster ovary (CHO) cell lines, various COScell lines, HeLa cells, L cells and myeloma cell lines. Preferably, thecells are nonhuman. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter, an enhancer, and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Queen et al., Immunol. Rev. 89:49, 1986. Preferred expression control sequences are promoters derivedfrom endogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. Co et al., J Immunol. 148: 1149, 1992.Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation, biolistics or viral-based transfectioncan be used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., Molecular Cloning). Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals. The vectors containing the DNA segments ofinterest can be transferred into the host cell by well known methods,depending on the type of cellular host.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding the gelsolin binding agent or can be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell that includes a gelsolin binding agent of the presentinvention, such as a prokaryotic or eukaryotic host cell in culture, canbe used to produce (i.e., express) recombinant gelsolin binding agent.In one embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding thegelsolin binding agent has been introduced) in a suitable medium suchthat the gelsolin binding agent is produced. In another embodiment, themethod further comprises the step of isolating the gelsolin bindingagent from the medium or the host cell. Once expressed, collections ofthe gelsolin binding agents, e.g., the anti-gelsolin antibodies or theanti-gelsolin antibody-related polypeptides are purified from culturemedia and host cells. The gelsolin binding agents can be purifiedaccording to standard procedures of the art, including HPLCpurification, column chromatography, gel electrophoresis and the like.In one embodiment, the gelsolin binding agent is produced in a hostorganism by the method of Boss et al., U.S. Pat. No. 4,816,397. Usually,anti-gelsolin antibody chains are expressed with signal sequences andare thus released to the culture media. However, if the anti-gelsolinantibody chains are not naturally secreted by host cells, theanti-gelsolin antibody chains can be released by treatment with milddetergent. Purification of recombinant polypeptides is well known in theart and include ammonium sulfate precipitation, affinity chromatographypurification technique, column chromatography, ion exchange purificationtechnique, gel electrophoresis and the like (see generally Scopes,Protein Purification (Springer-Verlag, N.Y., 1982).

Polynucleotides encoding gelsolin binding agents, e.g., theanti-gelsolin antibody coding sequences, can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal. See, e.g.,U.S. Pat. Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenesinclude coding sequences for light and/or heavy chains in operablelinkage with a promoter and enhancer from a mammary gland specific gene,such as casein or β-lactoglobulin. For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

Single Chain Antibodies. In one embodiment, the binding agent of theinvention is a single chain anti-gelsolin antibody. According to theinvention, techniques can be adapted for the production of single-chainantibodies specific to a gelsolin polypeptide (see, e.g., U.S. Pat. No.4,946,778). Examples of techniques which can be used to producesingle-chain Fvs and antibodies of the invention include those describedin U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods inEnzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci.USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040,1988.

Chimeric and Humanized Antibodies. In one embodiment, the binding agentof the invention is a chimeric anti-gelsolin antibody. In oneembodiment, the binding agent of the invention is a humanizedanti-gelsolin antibody. In one embodiment of the invention, the donorand acceptor antibodies are monoclonal antibodies from differentspecies. For example, the acceptor antibody is a human antibody (tominimize its antigenicity in a human), in which case the resultingCDR-grafted antibody is termed a “humanized” antibody.

Recombinant anti-gelsolin antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions, canbe made using standard recombinant DNA techniques, and are within thescope of the invention. For some uses, including in vivo use of thebinding agent of the invention in humans as well as use of these agentsin vitro detection assays, it is preferable to use chimeric, humanized,or human anti-gelsolin antibodies. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art. Such useful methods include, e.g., but are not limitedto, methods described in International Application No. PCT/US86/02269;U.S. Pat. No. 5,225,539; European Patent No. 184187, European Patent No.171496; European Patent No. 173494; PCT International Publication No. WO86/01533; U.S. Pat. Nos. 4,816,567; 5,225,539; European Patent No.125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987.Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol.139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura, et al., 1987. Cancer Res. 47: 999-1005; Wood, etal., 1985. Nature 314: 446-449; Shaw, et al., 1988. J. Natl. CancerInst. 80: 1553-1559); Morrison (1985) Science 229: 1202-1207; Oi, et al.(1986) BioTechniques 4: 214; Jones, et al., 1986. Nature 321: 552-525;Verhoeyan, et al., 1988. Science 239: 1534; Morrison, Science 229: 1202,1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol.Methods, 125: 191-202, 1989; U.S. Pat. No. 5,807,715; and Beidler, etal., 1988. Jr. Immunol. 141: 4053-4060. For example, antibodies can behumanized using a variety of techniques including CDR-grafting (EP 0 239400; WO 91/09967; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,859,205;6,248,516; EP460167), veneering or resurfacing (EP 0 592 106; EP 0 519596; Padlan E. A., Molecular Immunology, 28: 489-498, 1991; Studnicka etal., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). In oneembodiment, a cDNA encoding a murine anti-gelsolin monoclonal antibodyis digested with a restriction enzyme selected specifically to removethe sequence encoding the Fc constant region, and the equivalent portionof a cDNA encoding a human Fc constant region is substituted (seeRobinson et al., PCT/US86/02269; Akira et al., European PatentApplication, 184,187; Taniguchi, European Patent Application 171,496;Morrison et al., European Patent Application 173,494; Neuberger et al.,WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443;Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc.Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988)J. Natl. Cancer Inst. 80: 1553-1559); U.S. Pat. Nos. 6,180,370;6,300,064; 6,696,248; 6,706,484; 6,828,422.

In one embodiment, the present invention allows the construction ofhumanized anti-gelsolin antibodies that are unlikely to induce a humananti-mouse antibody (hereinafter referred to as “HAMA”) response, whilestill having an effective antibody effector function. As used herein,the terms “human” and “humanized”, in relation to antibodies, relate toany antibody which is expected to elicit a therapeutically tolerableweak immunogenic response in a human subject. In one embodiment, thepresent invention provides for a humanized gelsolin antibodies, heavyand light chain immunoglobulins.

CDR Antibodies. In one embodiment, the binding agent of the invention isan anti-gelsolin CDR antibody. Generally the donor and acceptorantibodies used to generate the anti-gelsolin CDR antibody aremonoclonal antibodies from different species; typically the acceptorantibody is a human antibody (to minimize its antigenicity in a human),in which case the resulting CDR-grafted antibody is termed a “humanized”antibody. The graft may be of a single CDR (or even a portion of asingle CDR) within a single V_(H) or V_(L) of the acceptor antibody, orcan be of multiple CDRs (or portions thereof) within one or both of theV_(H) and V_(L). Frequently all three CDRs in all variable domains ofthe acceptor antibody will be replaced with the corresponding donorCDRs, though one need replace only as many as necessary to permitadequate binding of the resulting CDR-grafted antibody to MetAp3.Methods for generating CDR-grafted and humanized antibodies are taughtby Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761; 5,693,762; andWinter U.S. Pat. No. 5,225,539; and EP 0682040. Methods useful toprepare V_(H) and V_(L) polypeptides are taught by Winter et al., U.S.Pat. Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP0368684; EPO451216; EP0120694.

After selecting suitable framework region candidates from the samefamily and/or the same family member, either or both the heavy and lightchain variable regions are produced by grafting the CDRs from theoriginating species into the hybrid framework regions. Assembly ofhybrid antibodies or hybrid antibody fragments having hybrid variablechain regions with regard to either of the above aspects can beaccomplished using conventional methods known to those skilled in theart. For example, DNA sequences encoding the hybrid variable domainsdescribed herein (i.e., frameworks based on the target species and CDRsfrom the originating species) can be produced by oligonucleotidesynthesis and/or PCR. The nucleic acid encoding CDR regions can also beisolated from the originating species antibodies using suitablerestriction enzymes and ligated into the target species framework byligating with suitable ligation enzymes. Alternatively, the frameworkregions of the variable chains of the originating species antibody canbe changed by site-directed mutagenesis.

Since the hybrids are constructed from choices among multiple candidatescorresponding to each framework region, there exist many combinations ofsequences which are amenable to construction in accordance with theprinciples described herein. Accordingly, libraries of hybrids can beassembled having members with different combinations of individualframework regions. Such libraries can be electronic database collectionsof sequences or physical collections of hybrids.

This process typically does not alter the acceptor antibody's FRsflanking the grafted CDRs. However, one skilled in the art can sometimesimprove antigen binding affinity of the resulting anti-gelsolin CDRgrafted antibody by replacing certain residues of a given FR to make theFR more similar to the corresponding FR of the donor antibody. Preferredlocations of the substitutions include amino acid residues adjacent tothe CDR, or which are capable of interacting with a CDR (see, e.g., U.S.Pat. No. 5,585,089, especially columns 12-16). Or one skilled in the artcan start with the donor FR and modify it to be more similar to theacceptor FR or a human consensus FR. Techniques for making thesemodifications are known in the art. Particularly if the resulting FRfits a human consensus FR for that position, or is at least 90% or moreidentical to such a consensus FR, doing so may not increase theantigenicity of the resulting modified anti-gelsolin CDR antibodysignificantly compared to the same antibody with a fully human FR.

Fusion Proteins. In one embodiment, the binding agent of the inventionis a fusion protein. The gelsolin binding agents of the presentinvention, when fused to a second protein, can be used as an antigenictag. Examples of domains that can be fused to polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but can occur through linker sequences. Moreover, fusion proteins of thepresent invention can also be engineered to improve characteristics ofthe gelsolin binding agent. For instance, a region of additional aminoacids, particularly charged amino acids, can be added to the N-terminusof the gelsolin binding agent to improve stability and persistenceduring purification from the host cell or subsequent handling andstorage. Also, peptide moieties can be added to the gelsolin bindingagent to facilitate purification. Such regions can be removed prior tofinal preparation of the gelsolin binding agent. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art. The gelsolin binding agent of the invention canbe fused to marker sequences, such as a peptide which facilitatespurification of the fused polypeptide. In preferred embodiments, themarker amino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824,1989, for instance, hexa-histidine provides for convenient purificationof the fusion protein. Another peptide tag useful for purification, the“HA” tag, corresponds to an epitope derived from the influenzahemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention. Also, thefusion protein can show an increased half-life in vivo.

Fusion proteins having disulfide-linked dimeric structures (due to theIgG) can be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

Similarly, EP-A-0 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, e.g., improvedpharmacokinetic properties. See EP-A 0232 262. Alternatively, deletingthe Fc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion can hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, e.g., human proteins, such as hIL-5,have been fused with Fc portions for the purpose of high-throughputscreening assays to identify antagonists of hIL-5. Bennett et al., J.Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem.,270: 9459-9471, 1995.

Labeled Gelsolin-Binding Agent. In one embodiment, the gelsolin bindingagent of the present invention is coupled with a label moiety, i.e.,detectable group. The particular label or detectable group conjugated tothe gelsolin binding agent of the invention is not a critical aspect ofthe invention, so long as it does not significantly interfere with thespecific binding of the gelsolin binding agent of the present inventionto the gelsolin polypeptide or the gelsolin-like polypeptide. Thedetectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and imaging, in general, most any label usefulin such methods can be applied to the present invention. Thus, a labelis any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texasred, rhodamine, and the like), radiolabels (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I,¹²¹I, ¹³¹I, ¹¹²In, ⁹⁹mTc), other imaging agents such as microbubbles forultrasound imaging), ¹⁸F, ¹¹C, ¹⁵O, (for Positron emission tomography),^(99m)TC, ¹¹¹In (for Single photon emission tomography), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and calorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, and the like)beads. Patents that described the use of such labels include U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;and 4,366,241, each incorporated herein by reference in their entiretyand for all purposes. See also Handbook of Fluorescent Probes andResearch Chemicals (6^(th) Ed., Molecular Probes, Inc., Eugene Oreg.).

The label can be coupled directly or indirectly to the desired componentof an assay according to methods well known in the art. As indicatedabove, a wide variety of labels can be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, e.g., biotin, thyroxine,and cortisol, it can be used in conjunction with the labeled,naturally-occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody, e.g., ananti-gelsolin antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds useful as labellingmoieties, include, but are not limited to, e.g., fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, andthe like. Chemiluminescent compounds useful as labelling moieties,include, but are not limited to, e.g., luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal-producing systems which can be used, see, U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels can bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels can be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies, e.g., the anti-gelsolin antibodies. In this case,antigen-coated particles are agglutinated by samples comprising thetarget antibodies. In this format, none of the components need belabeled and the presence of the target antibody is detected by simplevisual inspection.

Identifying and Characterizing the Gelsolin-Binding Agents of theInvention

Methods for identifying and/or screening the binding agents of theinvention. Methods useful to identify and screen the binding agents,e.g., anti-gelsolin antibodies and anti-gelsolin antibody-relatedpolypeptides, that possess the desired specificity to the gelsolinpolypeptide include any immunologically-mediated techniques known withinthe art. Components of an immune response can be detected in vitro byvarious methods that are well known to those of ordinary skill in theart. For example, (1) cytotoxic T lymphocytes can be incubated withradioactively labeled target cells and the lysis of these target cellsdetected by the release of radioactivity; (2) helper T lymphocytes canbe incubated with antigens and antigen presenting cells and thesynthesis and secretion of cytokines measured by standard methods(Windhagen A; et al., Immunity, 2: 373-80, 1995); (3) antigen presentingcells can be incubated with whole protein antigen and the presentationof that antigen on MHC detected by either T lymphocyte activation assaysor biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86:4230-4, 1989); (4) mast cells can be incubated with reagents thatcross-link their Fc-epsilon receptors and histamine release measured byenzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5)enzyme-linked immunosorbent assay (ELISA).

Similarly, products of an immune response in either a model organism(e.g., mouse) or a human subject can also be detected by various methodsthat are well known to those of ordinary skill in the art. For example,(1) the production of antibodies in response to vaccination can bereadily detected by standard methods currently used in clinicallaboratories, e.g., an ELISA; (2) the migration of immune cells to sitesof inflammation can be detected by scratching the surface of skin andplacing a sterile container to capture the migrating cells over scratchsite (Peters et al., Blood, 72: 1310-5, 1988); (3) the proliferation ofperipheral blood mononuclear cells in response to mitogens or mixedlymphocyte reaction can be measured using ³H-thymidine; (4) thephagocytic capacity of granulocytes, macrophages, and other phagocytesin PBMCs can be measured by placing PMBCs in wells together with labeledparticles (Peters et al., Blood, 72: 1310-5, 1988); and (5) thedifferentiation of immune system cells can be measured by labeling PBMCswith antibodies to CD molecules such as CD4 and CD8 and measuring thefraction of the PBMCs expressing these markers.

In one embodiment, gelsolin binding agents of the invention are selectedusing display of candidate binding agents on the surface of replicablegenetic packages. See, e.g., U.S. Pat. Nos. 5,514,548; 5,837,500;5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844306. Methods useful for producing/selecting a filamentous bacteriophageparticle containing a phagemid genome encoding for a binding moleculewith a desired specificity has been described. See, e.g., EP 774 511;U.S. Pat. Nos. 5,871,907; 5,969,108; 6,225,447; 6,291,650; 6,492,160.

In one embodiment, gelsolin binding agents of the invention are selectedusing display of candidate binding agents on the surface of a yeast hostcell. Methods useful for the isolation of scFv polypeptides by yeastsurface display have been described by Kieke et al., Protein Eng. 1997November; 10(11): 1303-10.

In one embodiment, gelsolin binding agents of the invention are selectedusing ribosome display. Methods useful for identifying ligands inpeptide libraries using ribosome display have been described byMattheakis et al., Proc: Natl. Acad. Sci. USA 91: 9022-26, 1994; andHanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

In one embodiment, gelsolin binding agents of the invention are selectedusing tRNA display of candidate binding agents. Methods useful for invitro selection of ligands using tRNA display have been described byMerryman et al., Chem. Biol., 9: 741-46, 2002.

In one embodiment, gelsolin binding agents of the invention are selectedusing RNA display. Methods useful for selecting peptides and proteinsusing RNA display libraries have been described by Roberts et al. Proc.Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBSLett., 414: 405-8, 1997. Methods useful for selecting peptides andproteins using unnatural RNA display libraries have been described byFrankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003.

In one embodiment, gelsolin binding agents of the invention areexpressed in the periplasm of gram negative bacteria and mixed withlabeled gelsolin polypeptide. See WO 02/34886. In clones expressingrecombinant polypeptides with affinity for the gelsolin polypeptide, theconcentration of the labeled gelsolin polypeptide bound to the bindingagents is increased and allows the cells to be isolated from the rest ofthe library as described in Harvey et al., Proc. Natl. Acad. Sci. 22:9193-98 2004 and U.S. Pat. Publication No. 2004/0058403.

After selection of the desired gelsolin binding agent, it iscontemplated that it can be produced in large volume by any techniqueknown to those skilled in the art, e.g., prokaryotic or eukaryotic cellexpression and the like. The gelsolin binding agents which are, e.g.,but not limited to, anti-gelsolin hybrid antibodies or fragments can beproduced by using conventional techniques to construct an expressionvector that encodes an antibody heavy chain in which the CDRs and, ifnecessary, a minimal portion of the variable region framework, that arerequired to retain original species antibody binding specificity (asengineered according to the techniques described herein) are derivedfrom the originating species antibody and the remainder of the antibodyis derived from a target species immunoglobulin which can be manipulatedas described herein, thereby producing a vector for the expression of ahybrid antibody heavy chain.

Measurement of Gelsolin Binding. In one embodiment, a gelsolin bindingassay refers to an assay format wherein a gelsolin polypeptide and agelsolin binding agent are mixed under conditions suitable for bindingbetween the gelsolin or gelsolin-like polypeptide and the gelsolinbinding agent and assessing the amount of binding between the gelsolinor gelsolin-like polypeptide and the gelsolin binding agent. The amountof binding is compared with a suitable control, which can be the amountof binding in the absence of the gelsolin polypeptide, the amount of thebinding in the presence of non-specific immunoglobulin composition, orboth. The amount of binding can be assessed by any suitable method.Binding assay methods include, e.g., ELISA, radioimmunoassays,scintillation proximity assays, fluorescence energy transfer assays,liquid chromatography, membrane filtration assays, and the like.Biophysical assays for the direct measurement of gelsolin polypeptidebinding to gelsolin binding agents are, e.g., nuclear magneticresonance, fluorescence, fluorescence polarization, surface plasmonresonance (BIACOR chips) and the like. Specific binding is determined bystandard assays known in the art, e.g., radioligand binding assays,ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectroscopyand the like. If the specific binding of a candidate gelsolin bindingagent is at least 1 percent greater than the binding observed in theabsence of the candidate gelsolin binding agent, the candidate gelsolinbinding agent is useful as a gelsolin binding agent of the invention.

Co-crystals of the gelsolin polypeptides and the gelsolin binding agentsare also provided by the present invention as a method of determiningmolecular interactions. Conditions suitable for binding between thegelsolin binding agent and a gelsolin polypeptide will depend on thecompound and its ligand and can be readily determined by one of ordinaryskill in the art.

Uses of the Gelsolin-Binding Agents of the Invention

General. The binding agents of the invention are useful in methods knownin the art relating to the localization and/or quantitation of agelsolin polypeptide (e.g., for use in measuring levels of the gelsolinpolypeptide within appropriate physiological samples, for use indiagnostic methods, for use in imaging the polypeptide, and the like).Binding agents of the invention are useful to isolate a gelsolinpolypeptide by standard techniques, such as affinity chromatography orimmunoprecipitation. A gelsolin binding agent of the invention canfacilitate the purification of natural immunoreactive gelsolinpolypeptides or gelsolin-like polypeptides from biological samples,e.g., mammalian sera or cells as well as recombinantly-producedimmunoreactive gelsolin polypeptides or gelsolin-like polypeptidesexpressed in a host system. Moreover, gelsolin binding agent can be usedto detect an immunoreactive gelsolin polypeptide or a gelsolin-likepolypeptide (e.g., in plasma, a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theimmunoreactive polypeptide. The gelsolin binding agents of the inventioncan be used diagnostically to monitor immunoreactive gelsolin and/orgelsolin-like polypeptide levels in tissue as part of a clinical testingprocedure, e.g., to determine the efficacy of a given treatment regimen.As noted above, the detection can be facilitated by coupling (i.e.,physically linking) the gelsolin binding agent of the invention to adetectable substance.

Detection of Gelsolin Polypeptide. An exemplary method for detecting thepresence or absence of a gelsolin polypeptide or a gelsolin-likepolypeptide in a biological sample involves obtaining a biologicalsample from a test subject and contacting the biological sample with agelsolin binding agent of the invention capable of detecting a gelsolinpolypeptide or a gelsolin-like polypeptide such that the presence of agelsolin polypeptide or a gelsolin-like polypeptide is detected in thebiological sample. An example of a gelsolin binding agent is an antibodyraised against SEQ ID NO.: 1 or a homlog or fragment thereof, capable ofbinding to a gelsolin polypeptide or a gelsolin-like polypeptide,preferably an antibody with a detectable label. The term “labeled”, withregard to the binding agent is intended to encompass direct labeling ofthe binding agent by coupling (i.e., physically linking) a detectablesubstance to the binding agent, as well as indirect labeling of thebinding agent by reactivity with another compound that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently-labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently-labeled streptavidin.

The detection method of the invention can be used to detect a gelsolinpolypeptide or a gelsolin-like polypeptide in a biological sample invitro as well as in vivo. In vitro techniques for detection of agelsolin polypeptide or a gelsolin-like polypeptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, radioimmunoassay, and immunofluorescence.Furthermore, in vivo techniques for detection of a gelsolin polypeptideor a gelsolin-like polypeptide include introducing into a subject alabeled gelsolin binding agent, e.g., an anti-gelsolin antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. In one embodiment, the biological sample containspolypeptide molecules from the test subject.

Immunoassay and Imaging. A gelsolin binding agent of the presentinvention can be used to assay gelsolin polypeptide levels orgelsolin-like polypeptide levels in a biological sample (e.g. humanplasma) using antibody-based techniques. For example, protein expressionin tissues can be studied with classical immunohistological methods.Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. etal., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methodsuseful for detecting protein gene expression include immunoassays, suchas the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, glucose oxidase, andradioisotopes or other radioactive agent, such as iodine (¹²⁵I, ¹²¹I,¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium (⁹⁹mTc), and fluorescent labels, such as fluorescein andrhodamine, and biotin.

In addition to assaying secreted gelsolin polypeptide levels orgelsolin-like polypeptide levels in a biological sample, secretedgelsolin polypeptide levels or gelsolin-like polypeptide levels can alsobe detected in vivo by imaging. A gelsolin binding agent, e.g., ananti-gelsolin antibody labels or markers for in vivo imaging of thegelsolin polypeptide levels or the gelsolin-like polypeptide includethose detectable by X-radiography, NMR or ESR. For X-radiography,suitable labels include radioisotopes such as barium or cesium, whichemit detectable radiation but are not overtly harmful to the subject.Suitable markers for NMR and ESR include those with a detectablecharacteristic spin, such as deuterium, which can be incorporated intothe gelsolin binding agent by labeling of nutrients for the relevantscFv clone.

A gelsolin binding agent which has been labeled with an appropriatedetectable imaging moiety, such as a radioisotope (e.g., ¹³¹I, ¹¹²In,⁹⁹mTc), a radio-opaque substance, or a material detectable by nuclearmagnetic resonance, is introduced (e.g., parenterally, subcutaneously,or intraperitoneally) into the subject. It will be understood in the artthat the size of the subject and the imaging system used will determinethe quantity of imaging moiety needed to produce diagnostic images. Inthe case of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of ⁹⁹mTc. The labeled gelsolin binding agent will thenpreferentially accumulate at the location of cells which contain thespecific target polypeptide. For example, in vivo tumor imaging isdescribed in S. W. Burchiel et al., Tumor Imaging: The RadiochemicalDetection of Cancer 13 (1982).

Thus, the invention provides a diagnostic method of a medical condition,which involves: (a) assaying the expression of a polypeptide bymeasuring binding of a gelsolin binding agent of the present inventionin cells or body fluid of an individual; (b) comparing the amount ofprotein with a standard, whereby an increase or decrease in the assayedpolypeptide compared to the standard level is indicative of a medicalcondition.

Affinity Purification. The gelsolin binding agents of the presentinvention may be used to purify immunoreacitve gelsolin (e.g., nativeplasma gelsolin) from a sample. In some embodiments, antibodies (e.g.GN3E9, GC1C10, and/or GF2D6) may be immobilized on a solid support.Examples of such solid supports include plastics such as polycarbonate,complex carbohydrates such as agarose and sepharose, acrylic resins andsuch as polyacrylamide and latex beads. Techniques for couplingantibodies to such solid supports are well known in the art (Weir etal., “Handbook of Experimental Immunology” 4th Ed., Blackwell ScientificPublications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth.Enzym. 34 Academic Press, N.Y. (1974)).

The simplest method to bind the antigen to the antibody-support matrixis to collect the beads in a column and pass the antigen solution downthe column. The efficiency of this method depends on the contact timebetween the immobilized antibody and the antigen, which can be extendedby using low flow rates. The immobilized antibody captures the antigenas it flows past. Alternatively, an antigen can be contacted with theantibody-support matrix by mixing the antigen solution with the support(e.g. beads) and rotating or rocking the slurry, allowing maximumcontact between the antigen and the immobilized antibody. After thebinding reaction has been completed, the slurry is passed into a columnfor collection of the beads. The beads are washed using a suitablewashing buffer and then the pure or substantially pure antigen iseluted.

An antibody or polypeptide of interest can be conjugated to a solidsupport, such as a bead. In addition, a first solid support such as abead can also be conjugated, if desired, to a second solid support,which can be a second bead or other support, by any suitable means,including those disclosed herein for conjugation of a polypeptide to asupport. Accordingly, any of the conjugation methods and means disclosedherein with reference to conjugation of a polypeptide to a solid supportcan also be applied for conjugation of a first support to a secondsupport, where the first and second solid support can be the same ordifferent.

Appropriate linkers, which can be cross-linking agents, for use forconjugating a polypeptide to a solid support include a variety of agentsthat can react with a functional group present on a surface of thesupport, or with the polypeptide, or both. Reagents useful ascross-linking agents include homo-bi-functional and, in particular,hetero-bi-functional reagents. Useful bi-functional cross-linking agentsinclude, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA,N-SPDP, SMCC and 6-HYNIC. A cross-linking agent can be selected toprovide a selectively cleavable bond between a polypeptide and the solidsupport. For example, a photolabile cross-linker, such as3-amino-(2-nitrophenyl)propionic acid can be employed as a means forcleaving a polypeptide from a solid support. (Brown et al., Mol. Divers,pp, 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996);and U.S. Pat. No. 5,643,722). Other cross-linking reagents arewell-known in the art. (See, e.g., Wong (1991), supra; and Hermanson(1996), supra).

An antibody or polypeptide can be immobilized on a solid support, suchas a bead, through a covalent amide bond formed between a carboxyl groupfunctionalized bead and the amino terminus of the polypeptide or,conversely, through a covalent amide bond formed between an amino groupfunctionalized bead and the carboxyl terminus of the polypeptide. Inaddition, a bi-functional trityl linker can be attached to the support,e.g, to the 4-nitrophenyl active ester on a resin, such as a Wang resin,through an amino group or a carboxyl group on the resin via an aminoresin. Using a bi-functional trityl approach, the solid support canrequire treatment with a volatile acid, such as formic acid ortrifluoracetic acid to ensure that the polypeptide is cleaved and can beremoved. In such a case, the polypeptide can be deposited as a beadlesspatch at the bottom of a well of a solid support or on the flat surfaceof a solid support. After addition of a matrix solution, the polypeptidecan be desorbed into a MS.

Hydrophobic trityl linkers can also be exploited as acid-labile linkersby using a volatile acid or an appropriate matrix solution, e.g., amatrix solution containing 3-HPA, to cleave an amino linked trityl groupfrom the polypeptide. Acid lability can also be changed. For example,trityl, monomethoxytrityl, dimethoxytrityl or trimethoxytrityl can bechanged to the appropriate p-substituted, or more acid-labiletritylamine derivatives, of the polypeptide, i.e., trityl ether andtritylamine bonds can be made to the polypeptide. Accordingly, apolypeptide can be removed from a hydrophobic linker, e.g., bydisrupting the hydrophobic attraction or by cleaving tritylether ortritylamine bonds under acidic conditions, including, if desired, undertypical MS conditions, where a matrix, such as 3-HPA acts as an acid.

Orthogonally cleavable linkers can also be useful for binding a firstsolid support, e.g., a bead to a second solid support, or for binding apolypeptide of interest to a solid support. Using such linkers, a firstsolid support, e.g., a bead, can be selectively cleaved from a secondsolid support, without cleaving the polypeptide from the support; thepolypeptide then can be cleaved from the bead at a later time. Forexample, a disulfide linker, which can be cleaved using a reducingagent, such as DTT, can be employed to bind a bead to a second solidsupport, and an acid cleavable bi-functional trityl group could be usedto immobilize a polypeptide to the support. As desired, the linkage ofthe polypeptide to the solid support can be cleaved first, e.g., leavingthe linkage between the first and second support intact. Trityl linkerscan provide a covalent or hydrophobic conjugation and, regardless of thenature of the conjugation, the trityl group is readily cleaved in acidicconditions.

For example, a bead can be bound to a second support through a linkinggroup which can be selected to have a length and a chemical nature suchthat high density binding of the beads to the solid support, or highdensity binding of the polypeptides to the beads, is promoted. Such alinking group can have, e.g., “tree-like” structure, thereby providing amultiplicity of functional groups per attachment site on a solidsupport. Examples of such linking group; include polylysine,polyglutamic acid, penta-erythrole and tris-hydroxy-aminomethane.

Noncovalent Binding Association. An antibody or polypeptide can beconjugated to a solid support, or a first solid support can also beconjugated to a second solid support, through a noncovalent interaction.For example, a magnetic bead made of a ferromagnetic material, which iscapable of being magnetized, can be attracted to a magnetic solidsupport, and can be released from the support by removal of the magneticfield. Alternatively, the solid support can be provided with an ionic orhydrophobic moiety, which can allow the interaction of an ionic orhydrophobic moiety, respectively, with a polypeptide, e.g., apolypeptide containing an attached trityl group or with a second solidsupport having hydrophobic character.

A solid support can also be provided with a member of a specific bindingpair and, therefore, can be conjugated to a polypeptide or a secondsolid support containing a complementary binding moiety. For example, abead coated with avidin or with streptavidin can be bound to apolypeptide having a biotin moiety incorporated therein, or to a secondsolid support coated with biotin or derivative of biotin, such asimino-biotin.

It should be recognized that any of the binding members disclosed hereinor otherwise known in the art can be reversed. Thus, biotin, e.g., canbe incorporated into either a polypeptide or a solid support and,conversely, avidin or other biotin binding moiety would be incorporatedinto the support or the polypeptide, respectively. Other specificbinding pairs contemplated for use herein include, but are not limitedto, hormones and their receptors, enzyme, and their substrates, anucleotide sequence and its complementary sequence, an antibody and theantigen to which it interacts specifically, and other such pairs knowsto those skilled in the art.

Diagnostic Uses of Gelsolin Binding Agents

General. The gelsolin binding compositions of the invention are usefulin diagnostic methods. As such, the present invention provides methodsusing the binding agents of the invention useful in the diagnosis ofgelsolin-related medical conditions in a subject. Binding agents of theinvention may be selected such that they have any level of epitopebinding specificity and very high binding affinity to the gelsolinpolypeptide. In general, the higher the binding affinity of an bindingagent the more stringent wash conditions can be performed in animmunoassay to remove nonspecifically bound material without removingtarget polypeptide. Accordingly, gelsolin binding agents of theinvention useful in diagnostic assays usually have binding affinities ofat least 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹²M⁻¹. Further, it is desirable thatgelsolin binding agents used as diagnostic reagents have a sufficientkinetic on-rate to reach equilibrium under standard conditions in atleast 12 h, preferably at least five (5) h and more preferably at leastone (1) hour.

Some methods of the invention employ polyclonal preparations ofanti-gelsolin antibodies and anti-gelsolin antibody compositions of theinvention as diagnostic reagents, and other methods employ monoclonalisolates. The use of polyclonal mixtures has a number of advantagescompared to compositions made of one monoclonal anti-gelsolin antibody.By binding to multiple sites on a gelsolin polypeptide, polyclonalanti-gelsolin antibodies or other polypeptides, one can generate astronger signal (for diagnostics) than a monoclonal that binds to asingle site on the gelsolin polypeptide or the gelsolin-likepolypeptide. Further, a polyclonal preparation can bind to numerousvariants of a prototypical target sequence (e.g., allelic variants,species variants, strain variants, drug-induced escape variants) whereasa monoclonal antibody can bind only to the prototypical sequence or anarrower range of variants thereto. However, monoclonal anti-gelsolinantibodies are advantageous for detecting a single antigen in thepresence or potential presence of closely related antigens.

In methods employing polyclonal human anti-gelsolin antibodies preparedin accordance with the methods described above, the preparationtypically contains an assortment of gelsolin binding agents, e.g.,antibodies, with different epitope specificities to the targetpolypeptide. In some methods employing monoclonal antibodies, it isdesirable to have two antibodies of different epitope bindingspecificities. A difference in epitope binding specificities can bedetermined by a competition binding assay.

Although gelsolin binding agents which are human antibodies can be usedas diagnostic reagents for any kind of sample, they are most useful asdiagnostic reagents for human biological samples. Gelsolin bindingagents can be used to detect a given gelsolin or gelsolin-likepolypeptide in a variety of standard assay formats. Such formats includeimmunoprecipitation, Western blotting, ELISA, radioimmunoassay, andimmunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual(Cold Spring Harbor Publications, New York, 1988); U.S. Pat. Nos.3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074, 3,791,932;3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517;3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and4,098,876. Biological samples can be obtained from any tissue or bodyfluid of a subject.

Immunometric or sandwich assays are a preferred format for thediagnostic methods of the present invention. See U.S. Pat. Nos.4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use onegelsolin binding agent, e.g., an anti-gelsolin antibody or a populationof anti-gelsolin antibodies immobilized to a solid phase, and anotheranti-gelsolin antibody or a population of anti-gelsolin antibodies insolution. Typically, the solution anti-gelsolin antibody or populationof anti-gelsolin antibodies is labeled. If an antibody population isused, the population can contain antibodies binding to different epitopespecificities within the target polypeptide. Accordingly, the samepopulation can be used for both solid phase and solution antibody. Ifanti-gelsolin monoclonal antibodies are used, first and second gelsolinmonoclonal antibodies having different binding specificities are usedfor the solid and solution phase. Solid phase (also referred to as“capture”) and solution (also referred to as “detection”) antibodies canbe contacted with target antigen in either order or simultaneously. Ifthe solid phase antibody is contacted first, the assay is referred to asbeing a forward assay. Conversely, if the solution antibody is contactedfirst, the assay is referred to as being a reverse assay. If the targetis contacted with both antibodies simultaneously, the assay is referredto as a simultaneous assay. After contacting the gelsolin polypeptidewith the anti-gelsolin antibody, a sample is incubated for a period thatusually varies from about 10 min to about 24 hr and is usually about 1hr. A wash step is then performed to remove components of the sample notspecifically bound to the anti-gelsolin antibody being used as adiagnostic reagent. When solid phase and solution antibodies are boundin separate steps, a wash can be performed after either or both bindingsteps. After washing, binding is quantified, typically by detecting alabel linked to the solid phase through binding of labeled solutionantibody. Usually for a given pair of antibodies or populations ofantibodies and given reaction conditions, a calibration curve isprepared from samples containing known concentrations of target antigen.Concentrations of the gelsolin polypeptide in samples being tested arethen read by interpolation from the calibration curve. Analyte can bemeasured either from the amount of labeled solution antibody bound atequilibrium or by kinetic measurements of bound labeled solutionantibody at a series of time points before equilibrium is reached. Theslope of such a curve is a measure of the concentration of the gelsolinpolypeptide in a sample

Suitable supports for use in the above methods include, e.g.,nitrocellulose membranes, nylon membranes, and derivatized nylonmembranes, and also particles, such as agarose, a dextran-based gel,dipsticks, particulates, microspheres, magnetic particles, test tubes,microtiter wells, SEPHADEX™ (Amersham Pharmacia Biotech, PiscatawayN.J.), and the like. Immobilization can be by absorption or by covalentattachment. Optionally, anti-gelsolin antibodies can be joined to alinker molecule, such as biotin for attachment to a surface boundlinker, such as avidin.

The invention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with gelsolin polypeptide expression or activity. Such assayscan be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with a gelsolin polypeptide. Furthermore,the methods of the present invention can also be used to assess whetheran individual expresses a gelsolin polypeptide or a polymorphic form ofthe gelsolin polypeptide in instances where a gelsolin binding agent ofthe present invention has greater affinity for the gelsolin polypeptidefor its polymorphic form (or vice versa).

The levels of certain polypeptides in a particular tissue (or in theblood) of a subject may be indicative of the toxicity, efficacy, rate ofclearance or rate of metabolism of a given drug when administered to thesubject. The methods described herein can also be used to determine thelevels of such polypeptide(s) (e.g. gelsolin or gelsolin-likepolypeptides) in subjects to aid in predicting the response of suchsubjects to these drugs. Another aspect of the invention providesmethods for determining gelsolin or gelsolin-like polypeptide expressionin an individual to thereby select appropriate therapeutic orprophylactic compounds for that individual.

The binding of a gelsolin binding agent of the invention to a gelsolinpolypeptide or a gelsolin-like polypeptide, e.g., can be utilized toidentify a subject having or at risk of developing a disorder associatedwith the gelsolin polypeptide or gelsolin-like polypeptide expression oractivity. Alternatively, the prognostic assays can be utilized toidentify a subject having or at risk for developing the disease ordisorder. Thus, the invention provides a method for identifying adisease or condition associated with an aberrant gelsolin polypeptide orgelsolin-like polypeptide expression or activity in which a test sampleis obtained from a subject and the gelsolin or gelsolin-like polypeptidedetected, wherein the presence of an alteration of gelsolin orgelsolin-like polypeptide is diagnostic for a subject having or at riskof developing a disease or condition associated with an aberrantgelsolin polypeptide or gelsolin-like polypeptide expression oractivity.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered a compound (e.g., anagonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with an aberrant gelsolin polypeptide or gelsolin-likepolypeptide expression or activity. For example, such methods can beused to determine whether a subject can be effectively treated with acompound affecting gelsolin polypeptide levels (e.g., a chemotherapeuticagent). Thus, the invention provides methods for determining whether asubject can be effectively treated with a compound for a disorder orcondition associated with an aberrant gelsolin polypeptide orgelsolin-like polypeptide expression or activity in which a test sampleis obtained and the gelsolin polypeptide or the gelsolin-likepolypeptide is detected using the gelsolin binding agent (e.g., whereinthe presence or absence of the gelsolin polypeptide or the gelsolin-likepolypeptide is diagnostic for a subject that can be administered thecompound to treat a disorder associated with an aberrant gelsolinpolypeptide or gelsolin-like polypeptide expression or activity).

The level of the gelsolin polypeptide or the gelsolin-like polypeptidein a blood or tissue sample obtained from a subject is determined andcompared with the level found in a blood sample or a sample from thesame tissue type obtained from an individual who is free of the disease.An underabundance (or overabundance) of the gelsolin polypeptide orgelsolin-like polypeptide in the sample obtained from the subjectsuspected of having the disease or condition affecting gelsolin levelscompared with the sample obtained from the healthy subject is indicativeof the gelsolin polypeptide or gelsolin-like polypeptide-associateddisease or condition in the subject being tested. Further testing may berequired to make a positive diagnosis.

There are a number of diseases in which the degree of underabundance (oroverabundance) of certain gelsolin polypeptide or gelsolin-likepolypeptide molecules known to be indicative of whether a subject withthe disease is likely to respond to a particular type of therapy ortreatment. Thus, the method of detecting a gelsolin polypeptide orgelsolin-like polypeptide in a sample can be used as a method ofprognosis, e.g., to evaluate the likelihood that the subject willrespond to the therapy or treatment. The level of the relevantprognostic polypeptide in a suitable tissue or blood sample from thesubject is determined and compared with a suitable control, e.g., thelevel in subjects with the same disease but who have responded favorablyto the treatment. The degree to which the prognostic polypeptide isunderexpressed in the sample compared with the control may be predictiveof likelihood that the subject will not respond favorably to thetreatment or therapy, e.g. tolerate chemotherapy treatment. The greaterthe overexpression (or underexpression) relative to the control, theless likely the subject will respond to the treatment. Examples ofconditions in which plasma gelsolin levels are decreased compared tocontrol subjects include, but are not limited to, septic shock, multipleorgan dysfunction syndrome, rheumatoid arthritis, trauma, stroke, heartinfarction, cancer, chemotherapy and radiation therapy, systemicautoimmune disease, and chronic hepatitis.

The methods described herein can be performed, e.g., by utilizingpre-packaged diagnostic kits comprising at least one probe reagent,e.g., gelsolin binding agent described herein, which can be convenientlyused, e.g., in clinical settings to diagnose subjects exhibitingsymptoms or family history of a disease or illness involving a gelsolinpolypeptide or gelsolin-like polypeptide. Furthermore, any cell type ortissue in which gelsolin polypeptide or gelsolin-like polypeptide isexpressed can be utilized in the prognostic assays described herein.

Correlating a Subject to a Standard Reference Population. To deduce acorrelation between clinical response to a treatment and a particularlevel of plasma gelsolin, it is necessary to obtain data on the clinicalresponses exhibited by a population of individuals who received thetreatment, i.e., a clinical population. This clinical data maybeobtained by retrospective analysis of the results of a clinicaltrial(s). Alternatively, the clinical data may be obtained by designingand carrying out one or more new clinical trials. The analysis ofclinical population data is useful to define a standard referencepopulation(s) which, in turn, are useful to classify subjects forclinical trial enrollment or for selection of therapeutic treatment. Ina preferred embodiment, the subjects included in the clinical populationhave been graded for the existence of the medical condition of interest.Grading of potential subjects can include, e.g., a standard physicalexam or one or more lab tests. Alternatively, grading of subjects caninclude use of a gene expression pattern. For example, plasma gelsolinlevel is a useful as grading criteria where there is a strongcorrelation between expression pattern and susceptibility or severity toa disease or condition. In one embodiment, a subject is classified orassigned to a particular group or class based on similarity between themeasured levels of a one or more biomarkers in the subject and the levelof the one or more biomarkers observed in a standard referencepopulation.

In one embodiment of the invention, a therapeutic treatment of interestis administered to each subject in a trial population, and eachsubject's response to the treatment is measured using one or morepredetermined criteria. It is contemplated that in many cases, the trialpopulation will exhibit a range of responses, and that the investigatorwill choose the number of responder groups (e.g., low, medium, high)made up by the various responses. In addition, the expression level of abiomarker (e.g. plasma gelsolin) is quantified, which may be done beforeand/or after administering the treatment. These results are thenanalyzed to determine if any observed variation in clinical responsebetween groups is statistically significant. Statistical analysismethods, which may be used, are described in L.D. Fisher & G. vanBelle,Biostatistics: A Methodology for the Health Sciences(Wiley-Interscience, New York, 1993).

The skilled artisan can construct a mathematical model that predictsclinical response as a function of expression level from the analysesdescribed above. The identification of an association between a clinicalresponse and an expression level for the biomarker may be the basis fordesigning a diagnostic method to determine those individuals who will orwill not respond to the treatment, or alternatively, will respond at alower level and thus may require more treatment, i.e., a greater dose ofa drug. The diagnostic method may take one of several forms: forexample, a ELISA or antibody-based test, a serological test, or aphysical exam measurement. The only requirement is that there be a goodcorrelation between the diagnostic test results and the underlyingcondition. In a preferred embodiment, this diagnostic method uses anantibody assay for serum gelsolin described above.

Predictive Medicine. The invention also pertains to the field ofpredictive medicine in which diagnostic assays, prognostic assays, andmonitoring clinical trials are used for prognostic (predictive) purposesto treat prophylactically a subject. Accordingly, one aspect of theinvention relates to diagnostic assays for determining plasma gelsolinlevels in the context of a biological sample (e.g., blood, serum, cells,tissue) to thereby determine whether an individual is afflicted with adisease or disorder, or is at risk of developing a disorder, associatedwith aberrant plasma gelsolin level.

Prognostic Assays. The binding of a prognostic compound to a biomarkermolecule, e.g., biomarker polypeptide or nucleic acid encoding abiomarker polypeptide, can be utilized to identify a subject having orat risk of developing a disorder associated with biomarker polypeptideexpression or activity (which are described above). A prognosticcompound is any compound which binds to or associates with a biomarkermolecule, including, but not limited to, e.g., anti-biomarkerpolypeptide antibody, small molecule, nucleic acid, polypeptide,oligosaccharide, lipid, or combination thereof. Alternatively, theprognostic assays can be utilized to identify a subject having or atrisk for developing the disease or disorder. Thus, the inventionprovides a method for identifying a disease or disorder associated withbiomarker expression or activity in which a test sample is obtained froma subject and prognostic compound binding or activity is detected,wherein the presence of an alteration of prognostic compound binding oractivity is diagnostic for a subject having, or at risk of developing, adisease or disorder associated with biomarker expression or activity. Asused herein, a “test sample” refers to a biological sample obtained froma subject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue, or isolated nucleic acid orpolypeptide derived therefrom.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered a compound (e.g., anagonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,small molecule, or other drug candidate) to treat a biomarker-associateddisease or disorder. As used herein, the administration of a compound toa subject or patient includes self-administration and the administrationby another. In one embodiment, the prognostic assays described hereinare used to determine if a subject will be responsive to a compound. Forexample, such methods can be used to determine whether a subject can beeffectively treated with a therapeutic compound for abiomarker-associated disorder (i.e., biomarker-associated medicalcondition). Thus, the invention provides methods for determining whethera subject can be effectively treated with a compound for a disorderassociated with biomarker expression or activity in which a test sampleis obtained and biomarker molecule is detected using prognostic compound(e.g., wherein the presence, or altered level of expression of, thebiomarker molecule compared with the level of expression of thebiomarker in a reference is diagnostic for a subject that can beadministered the compound to treat a biomarker-associated disorder.

In one embodiment, the level of the biomarker molecule in a blood ortissue sample obtained from a first subject is determined and comparedwith the level found in a blood sample or a sample from the same tissuetype obtained from an second subject free of the biomarker-associateddisease. An overabundance (or underabundance) of the biomarker moleculein the sample obtained from the first subject suspected of having thebiomarker associated disease compared with the sample obtained from thehealthy (second) subject is indicative of the biomarker-associateddisease in the subject being tested. Further testing may be required tomake a positive diagnosis.

In one embodiment, the level of the biomarker molecule (e.g., serumgelsolin) in a blood or tissue sample obtained from a subject at a firsttime point is determined and compared with the level found in a bloodsample or a sample from the same tissue type obtained from the subjectat a later time point. An overabundance (or underabundance) of thebiomarker molecule in the sample obtained from the subject at the firsttime point can be compared with the sample obtained from the subject atthe second time point wherein the decrease (underabundance) of thebiomarker level between the first time point compared with the biomarkerlevel at the second time point is indicative of a subject who is in needof gelsolin replacement therapy. Further testing may be required to makea positive diagnosis.

There are a number of diseases in which the degree of overexpression (orunderexpression) of certain biomarker molecules is known to beindicative of whether a subject with the disease is likely to respond toa particular type of therapy or treatment. Thus, the method of detectinga biomarker molecule in a sample can be used as a method of prognosis,e.g., to evaluate the likelihood that the subject will respond to thetherapy or treatment. Accordingly, in another embodiment, the level ofat least one biomarker molecules in a blood or tissue sample obtainedfrom a first subject is determined and compared with the level of the atleast one biomarker molecules found in a blood sample or a sample fromthe: same tissue type obtained from a second subject, or standardreference population, responsive to a compound, e.g., a therapeuticcompound of interest. Similarity in the level or pattern of expressionof the at least one biomarker molecules in a blood or tissue sampleobtained from a first subject compared with the level of the at leastone biomarker molecules found in a blood sample or a sample from thesame tissue type obtained from the second subject, or standard referencepopulation, indicates that the first subject will be responsive to thecompound, e.g., therapeutic compound of interest. That is, the level ofthe relevant biomarker in a suitable tissue or biological sample fromthe subject is determined and compared with a suitable control, e.g.,the level in subjects with the same disease but who have respondedfavorably to the treatment. The degree to which the biomarker isoverexpressed (or underexpressed) in the sample compared with thecontrol may be predictive of likelihood that the subject will notrespond favorably to the treatment or therapy (e.g. toleratechemotherapy). The greater the overexpression (or underexpression)relative to the control, the less likely the subject will respond to thetreatment.

There are a number of diseases in which the degree of overexpression (orunderexpression) of certain biomarker molecules, i.e.,biomarker-associated disease or medical condition, is known to beindicative of whether a subject will develop a disease. Thus, the methodof detecting a biomarker in a sample can be used as a method ofpredicting whether a subject will develop a disease. The level of a oneor more biomarkers in a suitable tissue or blood sample from a subjectat risk of developing the disease is determined and compared with asuitable control, e.g., the level in subjects who are not at risk ofdeveloping the disease. The degree to which the one or more biomarkersis overexpressed (or underexpressed) in the sample compared with thecontrol may be predictive of likelihood that the subject will developthe disease. The greater the overexpression (or underexpression)relative to the control, the more likely the subject will developmentthe disease.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe reagent,e.g., anti-gelsolin polypeptide antibody described herein, which can beconveniently used, e.g., in clinical setting to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga biomarker of the invention. Furthermore, any cell type or tissue inwhich a biomarker of the invention is expressed can be utilized in theprognostic assays described herein.

Monitoring Clinical Efficacy. In one embodiment, the present inventionprovides for monitoring the influence of agents (e.g., drugs, compounds,or small molecule) on the expression of gelsolin or gelsolin-likepolypeptides. Such assays can be applied in basic drug screening and inclinical trials. For example, the effectiveness of an agent to increase(or decrease) gelsolin or gelsolin-like polypeptide levels can bemonitored in clinical trials of subjects exhibiting decreased expressionof gelsolin. An agent that affects the expression of gelsolin orgelsolin-like polypeptides can be identified by administering the agentand observing a response. In this way, the expression pattern of thegelsolin or gelsolin-like polypeptide can serve as a marker, indicativeof the physiological response of the subject to the agent. Accordingly,this response state may be determined before, and at various pointsduring, treatment of the individual with the agent.

Subject Classification. Standard control levels of a gelsolin orgelsolin-like polypeptide are determined by measuring levels indifferent control groups. The control levels are then compared with themeasured level of a gelsolin or gelsolin-like polypeptide in a givensubject. The subject can be classified or assigned to a particular groupbased on how similar the measured levels were compared to the controllevels for a given group.

As one of skill in the art will understand, there will be a certaindegree of uncertainty involved in making this determination. Therefore,the standard deviations of the control group levels can be used to makea probabilistic determination and the method of this invention areapplicable over a wide range of probability-based genotype groupdeterminations. Thus, for example, and not by way of limitation, in oneembodiment, if the measured level of the gelsolin polypeptide fallswithin 2.5 standard deviations of the mean of any of the control groups,then that individual may be assigned to that group. In anotherembodiment if the measured level of the gene expression product fallswithin 2.0 standard deviations of the mean of any of the control groupsthen that individual may be assigned to that group. In still anotherembodiment, if the measured level of the gene expression product fallswithin 1.5 standard deviations of the mean of any of the control groupsthen that individual may be assigned to that group. In yet anotherembodiment, if the measured level of the gelsolin polypeptide is 1.0 orless standard deviations of the mean of any of the control groups levelsthen that individual may be assigned to that group.

Thus, this process allows determination, with various degrees ofprobability, which group a specific subject should be placed in, andsuch assignment would then determine the risk category into which theindividual should be placed.

Kits

Also within the scope of the invention are kits comprising the gelsolinbinding agent compositions (e.g., monoclonal antibodies) of theinvention and instructions for use. The kits are useful for detectingthe presence of a gelsolin polypeptide or a gelsolin-like polypeptide ina biological sample e.g., any body fluid including, but not limited to,e.g., serum, plasma, lymph, cystic fluid, urine, stool, cerebrospinalfluid, acitic fluid or blood and including biopsy samples of bodytissue. For example, the kit can comprise: one or more gelsolin bindingagents capable of binding a gelsolin polypeptide or a gelsolin-likepolypeptide in a biological sample (e.g. an antibody or antigen-bindingfragment thereof having the same antigen-binding specificity ofantibodies produced by a deposited cell line selected from the groupconsisting of: CGMCC Accession Nos: 2114, 2115, and 2116); means fordetermining the amount of the gelsolin polypeptide or gelsolin-likepolypeptide in the sample; and means for comparing the amount of thegelsolin polypeptide or the gelsolin-like polypeptide in the sample witha standard. One or more of the gelsolin binding agents may be labeled.The kit components, (e.g., reagents) can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect the gelsolin polypeptide or the gelsolin-like polypeptide.

For antibody-based kits, the kit can comprise, e.g., 1) a firstantibody, e.g., attached to a solid support, which binds to apolypeptide corresponding to a marker or the invention; and, optionally;2) a second, different antibody which binds to either the polypeptide orthe first antibody and is conjugated to a detectable label.

The kit can also comprise, e.g., a buffering agent, a preservative or aprotein-stabilizing agent. The kit can further comprise componentsnecessary for detecting the detectable-label, e.g., an enzyme or asubstrate. The kit can also contain a control sample or a series ofcontrol samples, which can be assayed and compared to the test sample.Each component of the kit can be enclosed within an individual containerand all of the various containers can be within a single package, alongwith instructions for interpreting the results of the assays performedusing the kit. The kits of the invention may contain a written producton or in the kit container. The written product describes how to use thereagents contained in the kit, e.g., to use the biomarkers of thepresent invention in determining a strategy for preventing or treating amedical condition in a subject. In several embodiments, the use of thereagents can be according to the methods of the invention.

Prophylactic and Therapeutic Use of Gelsolin Replacement Therapy

General. The gelsolin binding agents and methods of the presentinvention can be used in conjunction with gelsolin replacement therapy.Specifically, the invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with an aberrant gelsolinexpression or activity. The gelsolin binding agents and methods areused, for example, to ascertain the suitability of gelsolin replacementtherapy for a subject or monitor the efficacy of gelsolin replacementtherapy in a subject receiving such therapy. In one embodiment, atherapeutically effective amount of recombinant or purified, nativegelsolin compounds are administered so as to provide therapeuticbenefits against the secondary toxic effects of excessive extracellularactin. By “excessive” extracellular actin is meant an amount ofextracellular actin which exceeds the ability of the plasma proteins tobind and clear the actin from extracellular fluids without secondarytissue damage or toxic effects. By “secondary” tissue damage or toxiceffects is meant the tissue damage or toxic effects which occur tootherwise healthy tissues, organs, and the cells therein, due to thepresence of excessive extracellular actin in the plasma, usually as aresult of a “primary” tissue injury elsewhere in the body. While notwishing to be limited by theory, infusion of gelsolin, results in a)binding to actin monomers so as to prevent their condensation into actinfilaments, and/or b) cleavage of actin filaments to the monomeric state,and/or c) enhanced clearance of such actin complexed to actin-bindingproteins or fragments thereof from the circulation or extracellulartissue environment.

Optionally, the administration is made during the course of adjuncttherapy such as combined cycles of radiation, chemotherapeutictreatment, or administration of other cytoprotective or immunomodulatoryagent. As such the binding agents of the present invention and acompound useful in adjunct therapy may be administrated simultaneouslyand sequentially to a subject in need of administration thereof.

In one aspect, the invention provides a method for preventing, in asubject, a disease or condition associated with an aberrant gelsolinexpression or activity, by administering to the subject gelsolin.Administration of a prophylactic gelsolin binding agent can occur priorto the manifestation of symptoms characteristic of the aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. In therapeutic applications, gelsolin is administeredto a subject suspected of, or already suffering from, reduced serumgelsolin levels. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose.

Determination of the Biological Effect of the gelsolin-BindingAgent-Based Therapeutic. In various embodiments of the invention,suitable in vitro or in vivo assays are performed to determine theeffect of gelsolin replacement therapy and whether its administration isindicated for treatment of the affected tissue in a subject.

Typically, an effective amount of the compositions of the presentinvention, sufficient for achieving a therapeutic or prophylacticeffect, range from about 0.000001 mg per kilogram body weight per day toabout 10,000 mg per kilogram body weight per day. Preferably, the dosageranges are from about 0.0001 mg per kilogram body weight per day toabout 100 mg per kilogram body weight per day. For administration ofgelsolin, the dosage ranges from about 0.0001 to 100 mg/kg, and moreusually 0.01 to 5 mg/kg every week, every two weeks or every threeweeks, of the host body weight. For example dosages can be 1 mg/kg bodyweight or 10 mg/kg body weight every week, every two weeks or everythree weeks or within the range of 1-10 mg/kg every week, every twoweeks or every three weeks. In one embodiment, a single dosage ofantibody range from 0.1-10,000 micrograms per kg body weight. In oneembodiment, antibody concentrations in a carrier range from 0.2 to 2000micrograms per delivered milliliter. An exemplary treatment regimeentails administration once per every two weeks or once a month or onceevery 3 to 6 months. Gelsolin is usually administered on multipleoccasions. Intervals between single dosages can be daily, weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody in the subject. In some methods,dosage is adjusted to achieve a serum gelsolin concentration in thesubject of from about 75 μg/mL to about 125 μg/mL, 100 μg/mL to about150 μg/mL, from about 125 μg/mL to about 175 μg/mL, or from about 150μg/mL to about 200 μg/mL. Alternatively, gelsolin can be administered asa sustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the gelsolin binding agent in the subject. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some subjects continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the subject shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Toxicity. Preferably, an effective amount (e.g., dose) of gelsolindescribed herein will provide therapeutic benefit without causingsubstantial toxicity to the subject. Toxicity of the gelsolin describedherein can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀ (thedose lethal to 50% of the population) or the LD₁₀₀ (the dose lethal to100% of the population). The dose ratio between toxic and therapeuticeffect is the therapeutic index. The data obtained from these cellculture assays and animal studies can be used in formulating a dosagerange that is not toxic for use in human. The dosage of the gelsolindescribed herein lies preferably within a range of circulatingconcentrations that include the effective dose with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the subject's condition. See, e.g.,Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1(1975).

Formulations of Pharmaceutical Compositions. According to the methods ofthe present invention, the gelsolin can be incorporated intopharmaceutical compositions suitable for administration. Thepharmaceutical compositions generally comprise recombinant orsubstantially purified native gelsolin and a pharmaceutically-acceptablecarrier in a form suitable for administration to a subject.Pharmaceutically-acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions foradministering the antibody compositions (see, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18^(th) ed.,1990). The pharmaceutical compositions are generally formulated assterile, substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

The terms “pharmaceutically-acceptable,” “physiologically-tolerable,”and grammatical variations thereof, as they refer to compositions,carriers, diluents and reagents, are used interchangeably and representthat the materials are capable of administration to or upon a subjectwithout the production of undesirable physiological effects to a degreethat would prohibit administration of the composition. For example,“pharmaceutically-acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous. “Pharmaceutically-acceptable salts andesters” means salts and esters that are pharmaceutically-acceptable andhave the desired pharmacological properties. Such salts include saltsthat can be formed where acidic protons present in the gelsolin bindingagent are capable of reacting with inorganic or organic bases. Suitableinorganic salts include those formed with the alkali metals, e.g.,sodium and potassium, magnesium, calcium, and aluminum. Suitable organicsalts include those formed with organic bases such as the amine bases,e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Such salts also include acid additionsalts formed with inorganic acids (e.g., hydrochloric and hydrobromicacids) and organic acids (e.g., acetic acid, citric acid, maleic acid,and the alkane- and arene-sulfonic acids such as methanesulfonic acidand benzenesulfonic acid). Pharmaceutically-acceptable esters includeesters formed from carboxy, sulfonyloxy, and phosphonoxy groups presentin the gelsolin binding agent, e.g., C₁₋₆ alkyl esters. When there aretwo acidic groups present, a pharmaceutically-acceptable salt or estercan be a mono-acid-mono-salt or ester or a di-salt or ester; andsimilarly where there are more than two acidic groups present, some orall of such groups can be salified or esterified. The gelsolin bindingagent named in this invention can be present in unsalified orunesterified form, or in salified and/or esterified form, and the namingof such gelsolin binding agent is intended to include both the original(unsalified and unesterified) compound and itspharmaceutically-acceptable salts and esters. Also, certain gelsolinbinding agent named in this invention can be present in more than onestereoisomeric form, and the naming of such gelsolin binding agent isintended to include all single stereoisomers and all mixtures (whetherracemic or otherwise) of such stereoisomers. A person of ordinary skillin the art, would have no difficulty determining the appropriate timing,sequence and dosages of administration for particular drugs andcompositions of the present invention.

Preferred examples of such carriers or diluents include, but are notlimited to, water, saline, Ringer's solutions, dextrose solution, and 5%human serum albumin. Liposomes and non-aqueous vehicles such as fixedoils may also be used. The use of such media and compounds forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or compound is incompatible with thegelsolin binding agent, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. The gelsolincompositions of the present invention can be administered by parenteral,topical, intravenous, oral, subcutaneous, intraarterial, intradermal,transdermal, rectal, intracranial, intraperitoneal, intranasal;intramuscular route or as inhalants. The gelsolin can optionally beadministered in combination with other agents that are at least partlyeffective in treating various diseases including various actin- ormicrofilament-related diseases.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates, and compounds for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, e.g., water,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, e.g., by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalcompounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic compounds, e.g., sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition a compound which delays absorption, e.g., aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thegelsolin binding agent in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the binding agent into a sterile vehicle that containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The agents of this invention can be administered inthe form of a depot injection or implant preparation which can beformulated in such a manner as to permit a sustained or pulsatilerelease of the active ingredient.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the bindingagent can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding compounds, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating compound such as alginicacid, Primogel, or corn starch; a lubricant such as magnesium stearateor Sterotes; a glidant such as colloidal silicon dioxide; a sweeteningcompound such as sucrose or saccharin; or a flavoring compound such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the gelsolin binding agent aredelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, e.g., fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the gelsolin binding agent is formulated into ointments, salves, gels,or creams as generally known in the art.

The gelsolin an also be prepared as pharmaceutical compositions in theform of suppositories (e.g., with conventional suppository bases such ascocoa butter and other glycerides) or retention enemas for rectaldelivery.

In one embodiment, the gelsolin is prepared with carriers that willprotect the gelsolin against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically-acceptable carriers. These can beprepared according to methods known to those skilled in the art, e.g.,as described in U.S. Pat. No. 4,522,811.

Preparation of Recombinant Gelsolin. Most of the Discussion BelowPertains to production of gelsolin by culturing cells transformed with avector containing gelsolin nucleic acid and recovering the polypeptidefrom the cell culture. It is further envisioned that the gelsolin ofthis invention may be produced by purifying native gelsolin from plasmausing affinity purification (described above). The proteins and DNAsequences encoding the proteins may be produced using standard methods.Purification can also be accomplished using standard procedures such asisolating proteins using chromatography, using antibodies directedagainst the polypeptides, or by producing the polypeptides in a form inwhich they are fused to a moiety (or tag) that aids in purification andwhich can then be cleaved.

A polynucleotide encoding a gelsolin or gelsolin-like polypeptide (forexample, the polypeptide of SEQ ID NO.: 1) may be inserted into any ofthe many commercially available expression vectors using reagents andtechniques that are well known in the art. In preparing the recombinantexpression constructs, the various polynucleotides of the presentinvention may be inserted or substituted into a bacterialplasmid-vector. Any convenient plasmid may be employed, which will becharacterized by having a bacterial replication system, a marker whichallows for selection in a bacterium and generally one or more unique,conveniently located cloning sites. Numerous plasmids, also referred toas vectors, are available for transformation. Suitable vectors include,but are not limited to, the following: viral vectors, such as lambdavector system gt11, Charon 4, and plasmid vectors such as pBR322,pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,pKC37, pKC101, SV 40, pBluescript II SK+/−. or KS+/− (Stratagene, LaJolla, Calif.), and any derivatives thereof. Also suitable are yeastexpression vectors, which may be highly useful for cloning andexpression. Exemplary yeast plasmids include, without limitation, pPICZ,and pFLD. (Invitrogen, Carlsbad, Calif.). The selection of a vector willdepend on the preferred transformation technique and target host cells.

The nucleic acid molecule encoding gelsolin is inserted into a vector inthe 5′ to 3′ direction, such that the open reading frame is properlyoriented for the expression of the encoded protein under the control ofa promoter of choice. In this way, the gelsolin structural gene is saidto be “operably linked” to the promoter. Single or multiple nucleicacids may be inserted into an appropriate vector in this way, each underthe control of suitable promoters, to prepare a nucleic acid constructof the present invention.

Certain regulatory sequences may also be incorporated into theexpression constructs of the present invention. These includenon-transcribed regions of the vector, which interact with host cellularproteins to carry out transcription and translation. Such elements mayvary in their strength and specificity. Depending on the vector systemand host utilized, any number of suitable transcription and/ortranslation elements, including constitutive, inducible, and repressiblepromoters, as well as minimal 5′ promoter elements may be used.

A constitutive promoter is a promoter that directs constant expressionof a gene in a cell. Examples of some constitutive promoters that arewidely used for inducing expression of heterologous polynucleotidesinclude the ADH1 promoter for expression in yeast, those derived fromany of the several actin genes, which are known to be expressed in mosteukaryotic cell types, and the ubiquitin promoter, which is the promoterof a gene product known to accumulate in many cell types. Examples ofconstitutive promoters for use in mammalian cells include the RSVpromoter derived from Rous sarcoma virus, the CMV promoter derived fromcytomegalovirus, β-actin and other actin promoters, and the EF1αpromoter.

Also suitable as a promoter in the plasmids of the present invention isa promoter that allows for external control over the regulation of geneexpression. One way to regulate the amount and the timing of geneexpression is to use an inducible promoter. Unlike a constitutivepromoter, an inducible promoter is not always optimally active. Aninducible promoter is capable of directly or indirectly activatingtranscription of one or more DNA sequences or genes in response to aninducing agent (or inducer). Some inducible promoters are activated byphysical means, such as the heat shock promoter (HSP), which isactivated at certain temperatures. Other promoters are activated by achemical means, for example, IPTG. Other examples of inducible promotersinclude the metallothionine promoter, which is activated by heavy metalions, and hormone-responsive promoters, which are activated by treatmentof certain hormones. In the absence of an inducer, the nucleic acidsequences or genes under the control of the inducible promoter will notbe transcribed or will only be minimally transcribed. Promoters of thenucleic acid construct of the present invention may be either homologous(derived from the same species as the host cell) or heterologous(derived from a different species than the host cell).

Once the nucleic acid construct of the present invention has beenprepared, it may be incorporated into a host cell. This is carried outby transforming or transfecting a host or cell with a plasmid constructof the present invention, using standard procedures known in the art,such as described by Sambrook et al., Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor: Cold Spring Harbor LaboratoryPress, New York (2001). Suitable hosts and cells for the presentinvention include, without limitation, bacterial cells, virus, yeastcells, insect cells, plant cells, and mammalian cells, including humancells, as well as any other cell system that is suitable for producing arecombinant protein. Exemplary bacterial cells include, withoutlimitation, E. coli and Mycobacterium sp. Exemplary yeast hosts includewithout limitation, Pischia pastoris, Saccharomyces cerevisiae, andSchizosaccharomyces pombe. Methods of transformation or transfection mayresult in transient or stable expression of the genes of interestcontained in the plasmids. After transformation, the transformed hostcells can be selected and expanded in suitable culture. Transformedcells are first identified using a selection marker simultaneouslyintroduced into the host cells along with the nucleic acid construct ofthe present invention. Suitable markers include markers encoding forantibiotic resistance, such as resistance to kanamycin, gentamycin,ampicillin, hygromycin, streptomycin, spectinomycin, tetracycline,chloramphenicol, and the like. Any known antibiotic-resistance markercan be used to transform and select transformed host cells in accordancewith the present invention. Cells or tissues are grown on a selectionmedium containing an antibiotic, whereby generally only thosetransformants expressing the antibiotic resistance marker continue togrow. Additionally, or in the alternative, reporter genes, including,but not limited to, β-galactosidase, β-glucuronidase, luciferase, greenfluorescent protein (GFP) or enhanced green fluorescent protein (EGFP),may be used for selection of transformed cells. The selection markeremployed will depend on the target species.

Recombinant gelsolin may be purified using standard anion exchangechromatography. Oberley, R. E. et al., Am J Physiol Lung Cell MolPhysiol 287: L296-306 (2004).

To obtain the gelsolin protein, expression is induced if the codingsequences is under the control of an inducible promoter. To isolate theprotein, the host cell carrying an expression vector is propagated,homogenized, and the homogenate is centrifuged to remove bacterialdebris. The supernatant is then subjected to sequential ammonium sulfateprecipitation. The fraction containing the protein of the presentinvention is subjected to gel filtration in an appropriately sizeddextran or polyacrylamide column to separate the proteins. If necessary,the protein fraction may be further purified by HPLC. Alternativemethods of protein purification may be used as suitable. See J. E.Coligan et al., eds., Current Protocols in Protein Science (John Wiley &Sons, 2003). Upon obtaining the substantially purified recombinantprotein, the protein may be administered to a subject as describedherein.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of bindingagent calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the binding agent and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such gelsolin binding agent for thetreatment of a subject.

The following EXAMPLES are presented in order to more fully illustratethe preferred embodiments of the invention. These EXAMPLES should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLES

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1 Preparation of Gelsolin Antigens

1. Cloning of Gelsolin and Gelsolin Fragments

The cDNAs encoding the full-length, N-terminal, and C-terminal fragmentsof human gelsolin were cloned by the following RT-PCR method using:

a. Template

Total RNA was isolated from a HELA human cancer cell line. cDNA wassynthesized using a reverse transcription kit (Promega, Madison, Wis.,cat. no. A3500), according to the manufacturer's instructions. The cDNAwas used as a template for PCR.

b. PCR Primers

The following paired PCR primers for cloning full length human gelsolin,and the N- and C-terminal fragments are shown in Table 5:

TABLE 5 PCR Primers for Cloning Gelsolin Fragments. Gelsolin Amino acidProtein Primers SEQ ID NO range Full-length 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 5  13-782 (GF) 3′CTCGAGTCAGGCAGCCAGCTCAGCCAT SEQ ID NO.: 6 N-terminus 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 7  13-440 (GN) 3′TTACTCGAGTCCATATGTGGCAGGGTCCAC SEQ ID NO.: 8 C-terminus 5′CACCGGATCCGCCACATATGGACAGTTCT SEQ ID NO.: 9 440-782 (GC) 3′CTCGAGTCAGGCAGCCAGCTCAGCCAT SEQ ID NO.: 10

c. PCR Reaction

The following reagents were combined in the PCR reaction (100 μl finalvolume): template cDNA, 5 μl of total 33 μl reaction; 10 μmol of each 5′and 3′ primer; 10×PCR buffer, 10 μl; dNTPs (2.5 mM each), 4 μl; and Taqpolymerase (Promega), 5 units.

The PCR reaction was conducted as follows. The solution was first heatedat 94° C. for 2 min, followed by 40 cycles of 94° C. for 30 s, 52° C.for 1 min, and 72° C. for 3 min. The reaction was then incubated at 72°C. for 10 min for a final extension. The amplified DNA fragments, thusobtained, were separated on a 1% agarose gel containing 0.25 μg/mlethidium bromide. The PCR products were visualized using UV light andthe bands corresponding to the expected size of the amplificationproduct were recovered using the Gene Clean kit (BIO101, Irvine,Calif.).

d. Cloning of PCR Products

The DNA fragment was cloned using the TOPO100 expression Cloning Kit(Invitrogen, Carlsbad, Calif.). This was performed as follows. The DNAfragment recovered from the PCR reaction solution, together with 50 ngof TOPO vector which was provided with the cloning kit, was mixed with 1μl of 10× ligase reaction buffer (6 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride, 5 mM sodium chloride, 7 mM β-mercaptoethanol, 0.1 mM ATP, 2 mMDTT, 1 mM spermidine, and 0.1 mg/ml bovine serum albumin), to which 4units of T4 DNA ligase (1 μl) had been added. The total volume of themixture was adjusted to 10 μl with sterile deionized water, and theresulting ligase solution was incubated at 14° C. for 15 h. After thistime, 2 μl of the ligase reaction solution was added to 50 μl ofcompetent E. coli strain TOP10F, which was provided with the TA cloningkit and brought to competence in accordance with the instruction manual,and the resulting mixture was kept on ice for 30 min, then heated at 42°C. for 30 s, and then again chilled on ice for 5 min. Next, 500 μl ofmedium containing 2% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.05%(w/v) sodium chloride, 2.5 mM potassium chloride, 1 mM magnesiumchloride, and 20 mM glucose (hereinafter referred to as “SOC” medium)was added to the culture, and the mixture was incubated for 1 h at 37°C. with shaking. After this time, the culture was spread on an L-brothagar plate (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v)sodium chloride, 0.1% (w/v) glucose, and 0.6% (w/v) bacto-agar (Difco,Detroit, Mich.)), containing 100 μg/ml ampicillin Ampicillin resistantcolonies appearing on the plate were selected and scraped off with aplatinum transfer loop, and cultured in L-broth medium containing 100μg/ml ampicillin at 37° C., overnight, with shaking at 200 r.p.m. Afterincubation, the cells were harvested by centrifugation, from whichplasmid DNA was prepared by the alkali method as described in Sambrooket al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

2. Expression and Purification of Gelsolin Proteins

The cDNA encoding the full-length, the N-terminal or the C-terminalfragment of human gelsolin was inserted into the TOPO100 vector(Invitrogen, Carlsbad, Calif.). The resulting plasmids were transformedinto the E. coli strain BL21 (DE3), which was grown in LB media toexponential phases and induced with 0.4 mMisopropyl-1-thio-β-D-galactopyranoside for 3 h. Cells were pelleted bycentrifugation and the supernatant removed. The pelleted cells wereresuspended in lysis buffer (8 M urea, 20 mM Tris-HCl), and furtherdisrupted by sonication. This mixture was clarified by centrifugation(14,000×g for 15 min) and the supernatant recovered. The expressedrecombinant human gelsolin polypeptide was purified from the clarifiedsupernatant using a Ni-NTA superflow column (Qiagen, Valencia, Calif.)according to manufacturer's instructions. The purified gelsolin proteinpreparation was dialyzed against PBS at 4° C. overnight. The proteinconcentration was determined by BCA assay (Pierce, Woburn, Mass.), andaliquots were stored at −80° C. until use for immunization.

Purified gelsolin protein preparations (1 μg) were characterized byseparation on 10% SDS-PAGE and staining with Coomassie Blue. Afterdestain, the gel was scanned using an HP photographic scanner. Theresults are shown in FIG. 1 (Lane 1: molecular weight marker; Lane 2:purified human plasma gelsolin purchased from Cytoskeleton, Inc.(Denver, Colo.); Lane 3: recombinant full-length (“FL”) gelsolin; Lane4: recombinant N-terminal gelsolin fragment; Lane 5: recombinantC-terminal gelsolin fragment).

Example 2 Generation of Monoclonal Antibodies Against Human Gelsolin

1. Immunization

Female, Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) of 6-8 weeksof age, were immunized with one of: (1) native plasma gelsolin (NG,available from Cytoskeleton, Inc.); (2) recombinant full-length gelsolin(GF, amino acids 23-782 of SEQ ID NO.: 1), (3) recombinant N-terminalgelsolin (GN, amino acids 23-440 of SEQ ID NO.: 1), or (4) recombinantC-terminal gelsolin (GC, amino acids 440-782 of SEQ ID NO.: 1). For theinitial foot-pad immunization, 1 mg/mL of each immunogen was emulsifiedwith an equal volume of Freund's complete adjuvant (Difco, Detroit,Mich.). The mixture (100 μL) was injected into the foot pads of themice. Seven days later, the foot pads of the mice were injected with 100μL of each immunogen emulsified with an equal volume of adjuvant. Micewere further boosted weekly (for 3 weeks) with foot pad injection of 250μg immunogen in 100 μL PBS in combination with i.p. injection of 100 μgmurine recombinant B Lymphocyte Stimulator polypeptide (BLyS) in 0.5 mLPBS. Three days after the last injection, lymphocytes from the locallymph nodes of the immunized mice were collected.

2. Cell Fusion

Single cell suspension was prepared from lymph nodes, and mixed with NS1myeloma cells at a ratio of 2:1. The resulting mix was washed threetimes with PRMI-1640. One milliliter, 37° C. pre-warmed, of 50% (v/v)polyethylene glycol 1500 (Boehringer Mannheim, Basel, Switzerland) wasthen slowly added to the tube, while stirring the pellet using the tipof a pipette. Subsequently, 50 mL of serum-free RPMI medium, pre-warmedto 37° C., was slowly added. The resulting mix was then centrifuged, thesupernatant discarded and 50 mL of HAT medium containing 12% (v/v) FCSwas added while stirring gently with the tip of a pipette. Thesuspension was dispensed into 96-well cell culture microplates at 100μL/well. The plate was then incubated at 37° C. in an atmosphere of 5%(v/v) CO₂ for 7-10 days.

3. Screening of Monoclonal Antibody

The screening was conducted according to Table 6. ELISA plates werecoated with 1 μg/mL of the gelsolin immunogen protein at 4° C.overnight. Unbound gelsolin immunogen was rinsed from the wells bywashing three times with PBS. Non-specific binding sites in the wellswere blocked by incubating the wells with 3% (w/v) BSA PBS at roomtemperature for 1 h. The blocking buffer was removed prior to additionof hybridoma supernants without washing. One hundred microliters (100μL) of hybridoma culture supernatant was then added to appropriate wellsand the plate was incubated for 1 h at 37° C. to allow binding ofanti-gelsolin antibody. Unbound material was removed by rinsing thewells three times with PBS (5 min each). Bound anti-gelsolin antibodywas detected by incubating wells (30 min; 37° C.) with HRP-conjugatedanti-mouse IgG antibody (1:10,000). Unbound HRP-conjugated anti-mouseIgG antibody was removed by rinsing the wells three times with PBS (5min each). Anti-gelsolin antibody-HRP-conjugated anti-mouse IgG antibodycomplexes were measured using SureBlue TMB 1-Component MicrowellPeroxidase Substrate (KPL, Gaithersburg, Md.). Specifically, SureBlueTMB 1-Component Microwell Peroxidase Substrate (KPL, Gaithersburg, Md.)was added to the wells and the plate was incubated for 10 min to allowHRP-mediated conversion of the substrate. The enzymatic reaction wasstopped with the addition of 100 μl 2NH₂SO₄. The optical density of thesample wells was then measured at 450 nm/650 nm using an ELISA platereader.

TABLE 6 Screening Strategy Group Immunogen First Screening SecondScreening 1 Native Gelsolin (NG) NG GF GN GC 2 Full-Length RecombinantGF NG Gelsolin (FL) GN GC 3 N-terminal Recombinant GN NG Gelsolin (GN)GF GC 4 C-terminal Recombinant GC NG Gelsolin (GC) GF GN

A second confirmatory screening procedure was used to further define thebinding specificity of positive clones. All positive clones weresubjected to a secondary confirmatory ELISA screening (as detailedabove) using “secondary screening” polypeptides defined in Table 6. Theresults of the screening are shown in Table 7. The number of positiveclones compared with the total clones tested for each screen areindicated in Table 7. The primary screening tested 480 clones forbinding to each gelsolin immunogen. Those clones which were positive forbinding to the gelsolin immunogen were tested for binding to the othergelsolin immunogens of the study.

TABLE 7 Primary and Secondary Screening Results Screening ProteinImmunogen NG GF GN GC NG 34/480 2/34 1/34 1/34 GF 31/85  85/480 31/85 25/85  GN 4/37 19/37  37/480 0/37 GC 30/76  30/76  6/76 76/480

Three clones GN3E9, GC1C10, and GF2D6 were found to specifically bind tonative gelsolin (NG) and/or recombinant gelsolin (GF) as well as eitherN- or C-terminal fragments (GN or GC), with high binding affinity.Therefore, these clones were selected as exemplary antibodies useful inthe gelsolin detection methods of the invention (described below).

4. Cloning by Limiting Dilution

The original GN3E9, GC1C10, and GF2D6 hybridoma cells were diluted to0.3 cells per mL with RPMI-1640 containing 12% (v/v) FCS and cultured intwo 96-well plates in the presence of 10⁵ thymocytes of Balb/c mice asfeeder cells. Seven to ten days later (7-10 days), Culture supernatants(100 μL) were collected seven to ten days (7-10 days) later and antibodyproduction was determined by ELISA as described above. The positiveclones were subsequently subcloned three times by limiting dilution.

5. Analysis of Isotypes of Anti-Gelsolin Antibodies

The isotype of selected anti-gelsolin antibodies was determined by goatanti-murine isotype specific antibodies (SouthernBiotech, Birmingham,Ala.). Isotype of GN3E9 was determined as murine IgG2b kappa, and theisotype of GC1C10 and GF2D6 were determined as murine IgG1 kappa.

6. Purification of GN3E9, GC1C10, and GF2D6 Monoclonal Antibodies

GN3E9 was purified by affinity chromatography using Sepharose GL-4Baffinity purification medium (Pharmacia, Uppsala, Sweden). The GC andGF2D6 antibodies were purified by affinity chromatograpy using ProteinG-Sepharose CL-4B affinity purification medium (Pharmacia, Uppsala,Sweden). The culture supernatants were applied to the column with a flowrate of 2 ml per min. After the culture supernatant was passed throughthe column, it was washed with 50 ml PBS. The protein was eluted withelution buffer (0.1 M glycine (pH 2.4), 0.15 M NaCl). The opticaldensity of each eluted fraction (1 ml) was measured at OD280 nm. Thefractions with OD280>0.1 units were collected. After addition of 100 μlof neutralization buffer (1M Tris-HCl pH 8.5) to the fraction, theeluates were placed separately in dialysis tubing, and the eluatesdialyzed against 1 L of PBS (pH 7.5) at 4° C. The dialysis buffer waschanged twice. Each affinity purified antibody was concentrated to 1mg/ml, sterilized and stored at 4° C. until use.

Example 3 Characterization of Selected Gelsolin Binding Agents of thePresent Invention

Three selected antibodies (e.g., GN3E9, GF2D6, and GC1C10) as well ascommercial anti-human gelsolin antibody GS2C4 (Sigma Chemical Co., St.Louis, Mo.), were characterized using ELISA, western blot, andimmunoprecipitation techniques. Using the methods described below, itwould also be possible for the skilled artisan to generate and/orcompare additional gelsolin binding agents to the antibodies shown inthese examples.

1. Determination of Binding Characteristics of Gelsolin Binding Agentsby ELISA

Studies were performed to determine the binding characteristics ofselect gelsolin binding agents as detailed below. An ELISA plate wascoated with 1 μg/mL of native human plasma gelsolin purified from humanplasma (purchased from Cytoskeleton, Inc., Denver, Colo.), therecombinant full-length gelsolin (GF), the recombinant N-terminalfragment (GN), the recombinant C-terminal fragment (GC), or BSA as abackground control in PBS at 4° C. overnight. Unbound gelsolinpolypeptide was rinsed from the wells by washing the plate three timeswith PBS (5 min each). Non-specific binding sites in the wells wereblocked by incubating the wells with 3% (w/v) BSA PBS at roomtemperature for 1 h. A varying amount of antibody (0.001 μg/mL-10 μg/mL)was added at 37° C. for 30 min. Unbound antibody was removed from thewells by rinsing three times with PBS (5 min each). Bound anti-gelsolinantibody was detected by incubating wells (30 min; 37° C.) withHRP-conjugated anti-mouse IgG antibody (1:10,000, 100 μl). UnboundHRP-conjugated anti-mouse IgG antibody was removed by rinsing the wellsthree times with PBS (5 min each). Anti-gelsolin antibody-HRP-conjugatedanti-mouse IgG antibody complexes were measured using SureBlue TMB1-Component Microwell Peroxidase Substrate (KPL, Gaithersburg, Md.).Specifically, SureBlue TMB 1-Component Microwell Peroxidase Substrate(KPL, Gaithersburg, Md.) was added to the wells and the plate wasincubated for 10 min to allow HRP-mediated conversion of the substrate.The enzymatic reaction was stopped with the addition of 100 μl 2NH₂SO₄.The optical density of the sample wells was then measured at 450 nm/650nm using an ELISA plate reader.

A summary of the results of studies performed to determine the bindingcharacteristics of select gelsolin binding agents as detailed below areshown in FIG. 2. In panel A, GN3E9 showed a dose-dependent response tonative gelsolin, recombinant full-length, and recombinant N-terminalgelsolin fragment, but not to the C-terminal fragment or BSA, indicatingthat GN3E9 is an antibody directed against the N-terminal portion ofgelsolin. In panels B and C, GF2D6 and GC1C10, respectively, showed adose-dependent response to native gelsolin, recombinant full-length, andrecombinant C-terminal gelsolin fragment, but not to the N-terminalfragment or BSA, indicating that both GF2D6 and GC1C10 are antibodiesdirected against the C-terminal portion of gelsolin. The commercialanti-gelsolin antibody, GS2C4 (Panel D), showed a similar bindingspecificity to GF2D6 and GC1C 10, but the binding reactivity is muchweaker compared to GF2D6 and GC1C10. The GF2D6 antibody appearedsignificantly less sensitive to detect immunoreactive gelsolin as judgedby the lack of signal at 0.1 μg/ml concentration in GS2C4 (panel D)compared with signal observed for GN3E9 (panel A), GF2D6 (panel B), andGC1C10 (panel D) at the same concentration (0.1 μg/ml). As such, thegelsolin binding agent of the invention have the advantage of greatersensitivity to detect gelsolin and gelsolin related polypeptides whencompared to commercial anti-gelsolin antibody GS2C4. The highersensitivity of the gelsolin binding agents of the invention isadvantageous for use of these binding agents in methods of the presentinvention.

2. Western Blot Analysis

Western blot analysis technique was used to assess the bindingcharacteristics of the gelsolin binding agents of the invention inbiological samples. That is, to further determine the bindingspecificity of anti-gelsolin antibodies, western blot analysis of humanserum samples with anti-gelsolin antibodies was performed. The serumsamples of two normal human subjects (20 μl of 1:20 dilution of serum inPBS) was fractionated by 10% SDS-PAGE and western blotted ontonitrocellulose membrane using standard techniques. After blocking thewith electroblotted nitrocellulose membrane (blot) using 5% (w/v)non-fat milk at room temperature for 1 h. Each of four (4) replicateblots was probed with a purified anti-gelsolin antibody (1 μg/ml), i.e.,GN3E9; GC1C10; GF2D5; or GS2C4, at room temperature for 2 h. Unboundanti-gelsolin antibody was rinsed from the blots by washing with PBScontaining 0.02% Tween 20 at room temperature for 10 min with shaking.The bound anti-gelsolin antibody was detected by probing each blot withHRP-conjugated goat anti-murine IgG at room temperature for 1 h. UnboundHRP-conjugated goat anti-murine IgG was rinsed from the blots by washingwith PBS containing 0.02% Tween20 at room temperature for 10 min withshaking. The anti-gelsolin-HRP-conjugated goat anti-murine IgG complexeswere visualized using HRP-mediated chemiluminescence. Specifically, theblots were incubated with LumiGLO® Peroxidase Chemiluminescent Substrate(KPL, Gaithersburg, Md.) for 3 min, and exposed to X-ray films. Theresults are shown in FIG. 3. As shown in FIG. 3, GN3E9, GF2D6 and GS2C4,GC1C10 all bound a 90 kDa protein corresponding to the full-lengthplasma gelsolin. The GC1C10 antibody also bound other gelsolin-relatedpolypeptides present in human serum including a 50 kDa protein(s). The50 kDa gelsolin-like polypeptide is recognized by some antibodies whichare directed to an epitope in the C-terminus of full-length gelsolinpolypeptide.

3. Immunoprecipitation

To determine the ability of gelsolin binding agents of the invention toimmunoprecipitate the native form of plasma gelsolin, GN3E9, GF2D6,GC1C10 and GS2C4 antibodies were conjugated to CNBr-activated Sepharose4B (Amersham Pharmacia Biotech (Piscataway, N.J.), at a concentration of2 mg/ml beads. The conjugation procedure was performed according to themanufacturer's instructions. Briefly, the pre-activated beads (660 mg;equal to approximately 2 mL final bead volume) were suspended in 15volumes of 1 mM HCl and allowed to swell for 30 min. The beads were thenwashed with 15 gel volumes of cold (4° C.) 1 mM HCl followed by a washwith 15 volumes of coupling buffer (0.1M NaHCO₃ pH 8.3 containing 0.5MNaCl) to yield beads that are referred to as “washed gel”. Eachanti-gelsolin antibody (GC1C10; GF2D6; GN3E9; and GS2C4) was diluted incoupling buffer to 0.5 to 1.0 mg/ml and the pH was adjusted to pH 8.3.The washed gel was added to each anti-gelsolin antibody solution and themixtures were incubated overnight at 4° C. to yield “coupled gels”. Thecoupled gels were resuspended in 15 volumes of 1 M ethanolamine for 2-4h at room temperature to block unused activated chemical conjugationsites on the activated beads. The blocked gels were then washed 8 timesin 15 volumes with alternating 50 mM Tris, 1 M NaCl pH 8.0 and 50 mMglycine, 1 M NaCl pH 3.5 buffers followed by a final wash with 10 gelvolumes of PBS to remove any unbound material.

To measure the gelsolin binding agents ability to immunoprecipitatehuman gelsolin from human serum, one milliliter (1 mL) human serumsamples from normal subjects were incubated with 10 μl anti-gelsolinantibody-conjugated beads (i.e., GC1C10; GF2D6; GN3E9; or GS2C4conjugated beads) or blank beads (i.e., beads without anti-gelsolinantibody conjugated to them) as a control at room temperature for 2 h.Unbound material was rinsed from the anti-gelsolin antibody-conjugatedbeads or blank beads by pelleting the bead by centrifugation (14,000rpm, 3 min), removing the supernatant and then washing the pelletedbeads by resuspension in PBS. Following five (5) wash cycles, materialsbound to the anti-gelsolin antibody-conjugated beads or blank beads wasremoved under denaturing conditions by adding 40 μl SDS-PAGE loadingbuffer (SDS-PAGE loading buffer prepared by mixing 3× stock: 1M Tris-ClpH 6.8 2.4 ml; 20% SDS 3 ml; Glycerol (100%) 3 ml; B-mercaptoethanol 1.6ml; Bromophenol blue 0.006 g, 10 ml) to the pelleted beads and boilingthe sample for 5 min. The immunoprecipitated proteins were fractionatedon a 10% SDS-PAGE and visualized stained with Coomassie blue stain usingstandard techniques. The results are shown in FIG. 4. The lanes of theSDS-PAGE shown in FIG. 4 are as follows: Lane 1, blank beads (noantibody); lane 2, GC1C10; lane 3, GF2D6; lane 4, GN3E9; and lane 5,GS2C4. The anti-gelsolin antibodies of the invention (i.e., GC1C10,GF2D6 and GN3E9) shown in lanes 2-4, respectively, were able toimmunoprecipitate ˜90 kDa polypeptide consistent with the expectedmigration of full-length plasma gelsolin from human serum. In contrast,neither the commercial anti-gelsolin antibody, GS2C4 (Lane 5), nor theblank (no antibody control; Lane 1), exhibited the ability toprecipitate a detectable ˜90 kDa polypeptide from human serum sample. Assuch, the gelsolin binding agents of the invention tested in the presentstudies are distinct from the commercial anti-gelsolin antibody GS2C4 asthey can precipitate immunoreactive ˜90 kDa polypeptide consistent withthe expected migration of full-length gelsolin polypeptide from humanserum sample. The identity of this ˜90 kDa polypeptide was confirmed tobe full-length gelsolin polypeptide by mass spectroscopy analysis (SeeExample 4).

The ability of the select gelsolin binding agents of the inventiontested to immunoprecipitate is advantageous for use of these bindingagents in methods of the present invention. Specifically, gelsolinbinding agents of the invention which can precipitate immunoreactive ˜90kDa polypeptide (i.e., native gelsolin) are likely superior to bindnative gelsolin in biological sample and, consequently, prove to be moreuseful in the methods of the present invention when compared with otheranti-gelsolin antibody (e.g., GS2C4) which cannot precipitateimmunoreactive ˜90 kDa polypeptide from human serum sample under theconditions like those employed in the present studies.

Example 4 Characterization of the Immunoreactive Polypeptide Bound byGelsolin Binding Agents

To confirm that the 90 kDa protein immunoprecipitated by anti-gelsolinantibodies is human gelsolin, the protein band was cut from SDS-PAGE andthe contents subjected to analysis by mass spectroscopy at the NationalCenter of Biomedical Analysis (Beijing, China) using standard techniques(see Lewis et al., Identification of Viral Mutants by Mass Spectrometry,Proc Nat Acad Sci USA 95: 8596-8601 (1998)). The data are shown in Table8. A proteomics database (Swiss-Prot/TrEMBL) search indicated that the90 kDa protein is human gelsolin. That is, the mass spectroscopy patternobserved for the 90 kDa polypeptide which was immunoprecipitated by theanti-gelsolin antibodies of the invention matched the fragmentationpattern reported for human plasma gelsolin (Swiss-Prot/TrEMBL).Accordingly, the 90 kDa polypeptide which was immunoprecipitated by theanti-gelsolin antibodies of the invention was confirmed as human plasmagelsolin.

TABLE 8 Mass spectrometry analysis of the immunoprecipitated 90 kDaprotein. m/z Intens. 1 998.57 50843.46 2 1033.56 2703.13 3 1044.551646.67 4 1074.56 2661.98 5 1078.56 13005.95 6 1118.55 3156.02 7 1126.653472.77 8 1179.65 1752.49 9 1208.74 2901.21 10 1210.76 9589.77 111231.78 2089.52 12 1234.68 6770.12 13 1254.76 130596.74 14 1260.752464.65 15 1275.78 178815.66 16 1279.76 1859.19 17 1293.70 3749.91 181308.71 1859.32 19 1315.74 5299.92 20 1320.64 3175.49 21 1349.70 6692.3622 1434.84 1473.61 23 1475.82 4269.13 24 1493.80 2938.58 25 1526.8413178.06 26 1538.84 3921.98 27 1542.83 3212.21 28 1554.85 2676.44 291573.80 2077.29 30 1599.87 2777.20 31 1639.78 1360.55 32 1700.92 2838.9133 1722.91 17508.64 34 1736.86 1608.93 35 1753.97 1432.72 36 1813.001890.69 37 1826.96 2585.75 38 1830.03 7243.12 39 1837.97 10582.00 401849.95 17521.04 41 1911.03 2400.38 42 1936.98 6510.56 43 1955.051325.40 44 1998.12 2783.94 45 2039.65 4259.79 46 2079.14 62184.58 472085.15 1201.01 48 2095.13 9613.80 49 2150.15 2518.35 50 2163.13 3361.5751 2272.16 13436.04 52 2294.14 897.53 53 2326.30 1187.02 54 2345.181417.40 55 2387.22 1715.26 56 2464.28 2647.65 57 2562.43 546.82 582669.34 1409.68 59 2687.35 1054.55 60 2706.46 3530.63 61 2764.51 682.4862 2771.42 832.80 63 2843.46 1059.69 64 2873.38 3997.29 65 2889.36558.77 66 3029.49 648.16 67 3570.92 306.74 68 3958.23 362.16 69 4087.37124.00 70 4273.48 78.00

Example 5 Characterization of Epitopes of Select Gelsolin Binding Agentsof the Invention

The epitope recognized by the anti-gelsolin antibodies of the inventionwas mapped to a 50-residue region of human gelsolin using the truncatedgelsolin polypeptides of distinct but overlapping amino acid sequencesof human gelsolin polypeptide.

1. Expression of Truncated Gelsolin Polypeptides

The epitopes recognized by GN3E9, GC1C10, and GF2D6 antibodies, as wellas the commercially available anti-gelsolin GS2C4 antibody (SigmaChemical Co., St. Louis, Mo., USA) were determined using a panel of thetruncated recombinant human gelsolin proteins.

a. Design of Truncated Human Gelsolin Proteins

Total RNA was isolated from a HELA human cancer cell line. cDNA wassynthesized using a reverse transcription kit (Promega, Madison, Wis.,cat. no. A3500), according to the manufacturer's instructions. The cDNAwas used as a template for PCR. The primers used for obtaining the cDNAclones encoding the truncated gelsolin proteins and the correspondingamino acid sequences of the truncated proteins are summarized in Table9.

TABLE 9 Cloning of the truncated human gelsolin for epitope mappingProtein Peptide Primers SEQ ID NO.: sequence Epitope GN1 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 11 aa13-aa466 aa416-aa466 3′GGATCCCTATCCATATGTGGCAGGGTC SEQ ID NO.: 12 GN2 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 13 aa13-aa416 aa366-aa416 3′GGATCCCTAGTTGGCGATATGGCTGGA SEQ ID NO.: 14 GN3 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 15 aa13-aa366 aa316-aa366 3′GGATCCCTAGATGAAGTCAGAGGCTGT SEQ ID NO.: 16 GN4 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 17 aal3-aa316 aa266-aa316 3′GGATCCCTAAGCCACGAGGGAGACGG SEQ ID NO.: 18 GN5 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 19 aa13-aa266 aa216-aa266 3′GGATCCCTACTCAGTGCCCTCCTCAGA SEQ ID NO.: 20 GN6 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 21 aa13-aa216 aa166-aa216 3′GGATCCCTAGAAGCAGTCGCCATTGTT SEQ ID NO.: 22 GN7 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 23 aa13-aa166 aa116-aa166 3′GGATCCCTACTTCAGGCCAGACTTGAA SEQ ID NO.: 24 GN8 5′CACCGGATCCCTGCTTTGCGCGCTGTCCCTG SEQ ID NO.: 25 aa13-aa116 aa66-aa116 3′GGATCCCTACAGCCAGTAGTGGAGGTC SEQ ID NO.: 26 GC1 5′CACCGGATCCGCCACATATGGACAGTTCT SEQ ID NO.: 27 aa467-aa782 aa732-aa782 3′GGATCCCTATCAGGCAGCCAGCTCAGC SEQ ID NO.: 28 GC2 5′CACCGGATCCGCCACATATGGACAGTTCT SEQ ID NO.: 29 aa467-aa732 aa682-aa732 3′GGATCCCTACGTCTCGATGTACCGCTT SEQ ID NO.: 30 GC3 5′CACCGGATCCGCCACATATGGACAGTTCT SEQ ID NO.: 31 aa467-aa682 aa632-aa682 3′GGATCCCTACTCTTCGATCACAAAACG SEQ ID NO.: 32 GC4 5′CACCGGATCCGCCACATATGGACAGTTCT SEQ ID NO.: 33 aa467-aa632 aa582-aa632 3′GGATCCCTATGCCACCTGCACAGGTTG SEQ ID NO.: 34 GC5 5′CACCGGATCCGCCACATATGGACAGTTCT SEQ ID NO.: 35 aa467-aa582 aa532-aa582 3′GGATCCCTAAGGCAATACCTCAACAGC SEQ ID NO.: 36

b. PCR Reaction

DNA encoding each human gelsolin peptide summarized in Table 5, Table 6or Table 9 was amplified from a composition comprising human nucleicacid as template. Briefly, the following reagents were combined in thePCR reaction (100 μA final volume): template cDNA, 5 μl of total 33 μlreaction; 10 pmol of the appropriate 5′ and 3′ primer pair (see Table 5or Table 9); 10×PCR buffer, 10 μl; dNTPs (2.5 mM each), 4 μl; and Taqpolymerase (Promega), 5 units.

The PCR reaction was conducted as follows. The solution was first heatedat 94° C. for 2 min, followed by 40 cycles of 94° C. for 30 s, 52° C.for 1 min, and 72° C. for 3 min. The reaction was then incubated at 72°C. for 10 min for a final extension. The amplified DNA fragments wereseparated on a 1% agarose gel containing 0.25 μg/ml ethidium bromide andvisualized using IN light. The bands corresponding to the expected sizeof the amplification product were recovered using the Gene Clean kit(BIO101, Irvine, Calif.). The identity of all PCR products was confirmedby sequence analysis (see below).

c. Cloning of PCR Products Encoding Truncated Human Gelsolin Polypeptide

Each DNA fragment encoding truncated human gelsolin polypeptide (Table5, Table 6 and Table 9) was cloned using the TOPO Expression Cloning Kit(Invitrogen, CA). Briefly, the DNA fragment recovered from the PCRreaction solution, together with 50 ng of TOPO expression vector (TOPOExpression Cloning kit), was mixed with 1 μl of 10× ligase reactionbuffer (6 mM Tris-HCl, pH 7.5, 6 mM magnesium chloride, 5 mM sodiumchloride, 7 mM β-mercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 mM spermidine,and 0.1 mg/ml BSA), to which 4 units of T4 DNA ligase (1 μl) had beenadded. The total volume of the mixture was adjusted to 10 μl withsterile deionized water, and the resulting ligase solution was incubatedat 14° C. for 15 h. Following incubation, 2 μl of the ligase reactionsolution was added to 50 p. 1 of competent E. coli strain TOP10F (TOPOExpression Cloning Kit) and brought to competence in accordance with themanufacturer's instructions. The resulting mixture was kept on ice for30 min, then treated at 42° C. for 30 s, and then again chilled on icefor 5 min. Next, 500 μl of medium containing 2% (v/v) tryptone, 0.5%(w/v) yeast extract, 0.05% (w/v) sodium chloride, 2.5 mM potassiumchloride, 1 mM magnesium chloride, and 20 mM glucose (hereinafterreferred to as “SOC” medium) was added to the culture, and the mixturewas incubated for 1 h at 37° C. with shaking. After this time, theculture was spread on an L-broth agar plate (1% (v/v) tryptone, 0.5%(w/v) yeast extract, 0.5% (w/v) sodium chloride, 0.1% (w/v) glucose, and0.6% (w/v) bacto-agar (Difco, Detroit, Mich.)), containing 100 μg/mlampicillin. Ampicillin resistant colonies appearing on the plate wereselected and scraped off with a platinum transfer loop and cultured inL-broth medium containing 100 μg/ml ampicillin at 37° C., overnight,with shaking at 200 r.p.m. After incubation, the cells were harvested bycentrifugation, from which plasmid DNA was prepared by the alkali method(Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).Five clones from each truncated clone were sequenced to identify clonesencoding predicted polypeptide sequence of each truncated gelsolinpolypeptide. A single clone verified to encode the predicted polypeptidesequence of each truncated gelsolin polypeptide was then selected forexpression in E. coli expression host.

d. Expression and Purification of Gelsolin Proteins

The cDNA encoding the full-length, the N-terminal or the C-terminalfragment of human gelsolin (Table 5 or Table 6) or the gelsolinpolypeptide truncations shown in (Table 9) were each inserted intoseparate TOPO100 vectors (Invitrogen). The resulting plasmids weretransformed into the Escherichia coli strain BL21 (DE3), which was grownin LB media to exponential phase and induced with 0.4 mMisopropyl-1-thio-β-D-galactopyranoside for 3 h. Cells were pelleted bycentrifugation and the supernatant removed. The pelleted cells wereresuspended in lysis buffer (8 M urea, 20 mM Tris-HCl), and furtherdisrupted by sonication. This mixture was clarified by centrifugation(14,000×g for 15 min) and the supernatant recovered. The expressedrecombinant human gelsolin polypeptide was purified from the clarifiedsupernatant using a Ni-NTA superflow column (Qiagen, Valencia, Calif.)according to manufacturer's instructions. The purified gelsolin proteinpreparation was dialyzed against PBS at 4° C. overnight. The proteinconcentration was determined by BCA assay (Pierce, Woburn, Mass.), andaliquots were stored at −80° C. until use.

2. Epitope Mapping by ELISA with Truncated Human Gelsolin Proteins.

An ELISA plate (96 well; BD Biosciences, CA) was coated (4° C.overnight) with 1 μg/ml of each truncated gelsolin protein as listed inTables 10 and 11. Unbound truncated gelsolin polypeptide was rinsed fromthe plate by washing the wells three times with PBS. The plate was thenblocked with 3% (w/v) BSA PBS at room temperature for 1 h. Test antibody(at 1 μg/mL final concentration) was added to appropriate wells andincubated at 37° C. for 1 h. The unbound antibodies were removed fromthe wells by washing the plate three times with PBS. Bound anti-gelsolinantibody was detected by incubating wells (30 min; 37° C.) withHRP-conjugated anti-mouse IgG antibody (diluted 1:10,000;SouthernBiotech, Birmingham, Ala.)). Unbound HRP-conjugated anti-mouseIgG antibody was removed by rinsing the wells three times with PBS (5min each). Anti-gelsolin antibody-HRP-conjugated anti-mouse IgG antibodycomplexes were measured using SureBlue TMB 1-Component MicrowellPeroxidase Substrate (KPL, Gaithersburg, Md.). Specifically, SureBlueTMB 1-Component Microwell Peroxidase Substrate (KPL, Gaithersburg, Md.)was added to the wells and the plate was incubated for 10 min to allowHRP-mediated conversion of the substrate. The enzymatic reaction wasstopped with the addition of 100 μl 2N H₂SO₄. The optical density of thesample wells was then measured at 450 nm/650 nm using an ELISA platereader.

A total of 9 anti-gelsolin antibody clones which produce monoclonalantibody directed against the N-terminal fragment of human gelsolin wereexamined with a panel of the N-terminal human gelsolin truncatedpolypeptides (GN1-GN8). Similarly, 16 anti-gelsolin antibody cloneswhich produce monoclonal antibody directed against the C-terminalfragment of human gelsolin were examined with a panel of the C-terminalhuman gelsolin truncated polypeptides (GC1-GC6). The epitope frequencyof the N-terminal and C-terminal specific antibodies is shown in Tables10 and 11, respectively.

TABLE 10 Epitope frequency of the N-terminus specific anti-gelsolinantibodies. Peptide Sequence Epitope Frequency Representative GN1aa13-aa466 aa416-aa466 0/9 GN2 aa13-aa416 aa366-aa416 0/9 GN3 aa13-aa366aa316-aa366 4/9 GN3E9 GN4 aa13-aa316 aa266-aa316 0/9 GN5 aa13-aa266aa216-aa266 0/9 GN6 aa13-aa216 aa166-aa216 0/9 GN7 aa13-aa166aa116-aa166 0/9 GN8 aa13-aa116  aa66-aa116 5/9 GF5A3

As shown in Table 10, four (4) of the nine (9) tested anti-gelsolinantibody clones which produce monoclonal antibody directed against theN-terminal fragment of human gelsolin were directed to region GN3. Onesuch anti-human gelsolin antibody is GN3E9. The remaining five (5) ofthe nine (9) tested anti-gelsolin antibody clones which producemonoclonal antibody directed against the N-terminal fragment of humangelsolin were directed to region GN8. One such anti-human gelsolinantibody is GN5A3.

TABLE 11 Epitope frequency of the C-terminus specific anti-gelsolinantibodies. Peptide Sequence Epitope Frequency Representative GC1aa467-aa782 aa732-aa782 0/16 GC2 aa467-aa732 aa682-aa732 0/16 GC3aa467-aa682 aa632-aa682 11/16  GC1C10, GP2D6 Sigma GS2C4 GC4 aa467-aa632aa582-aa632 0/16 GC5 aa467-aa582 aa532-aa582 0/16 GC6 aa467-aa532aa482-aa532 5/16 GC5C1

As shown in Table 11, eleven (11) of the sixteen (16) testedanti-gelsolin antibody commercially available GS2C4 antibody cloneswhich produce monoclonal antibody directed against the C-terminalfragment of human gelsolin were directed to region GC3. Three suchanti-human gelsolin antibodies are GCICIO, GF2D6 and the commerciallyavailable GS2C4 antibody. The remaining five (5) of the sixteen (16)tested anti-gelsolin antibody clones which produce monoclonal antibodydirected against the C-terminal fragment of human gelsolin were directedto region GC6. One such anti-human gelsolin antibody is GC5C1.

3. Fine Mapping of Epitopes by Analysis of Cross-Reactivity and HomologyAmong Species

The high level of homology of gelsolin polypeptides among differentspecies allowed further mapping of the gelsolin epitopes bound bygelsolin binding agents of the invention by examining cross-reactivityof these binding agents with gelsolin immunoreactive polypeptideexpressed in different species.

a. Immunoprecipitation

To determine the ability of anti-gelsolin antibodies of the invention toimmunoprecipitate the native form of plasma gelsolin from variousspecies, GN3E9, GF2D6 and GC1C10 were conjugated to Sepharose 4B at aconcentration of 2 mg/ml beads as described in Example 3 above. One mLserum samples (1 mL) from select mammalian species were incubated with10 μl antibody-conjugated beads or blank beads (control) at roomtemperature for 2 h. Unbound material was rinsed from the anti-gelsolinantibody-conjugated beads or blank beads by pelleting the bead bycentrifugation (14,000 rpm, 3 min), removing the supernatant and thenwashing the pelleted beads by resuspension in PBS. Following five (5)wash cycles, materials bound to the anti-gelsolin antibody-conjugatedbeads or blank beads was removed under denaturing conditions by adding40 μl SDS-PAGE loading buffer (SDS-PAGE loading buffer prepared bymixing 3× stock: 1M Tris-Cl pH 6.8 2.4 ml; 20% SDS 3 ml; Glycerol (100%)3 ml; B-mercaptoethanol 1.6 ml; Bromophenol blue 0.006 g, 10 ml) to thepelleted beads and boiling the sample for 5 min. The immunoprecipitatedproteins were fractionated on a 10% SDS-PAGE and visualized stained withCoomassie blue stain using standard techniques. Results are shown inFIG. 5A-5C.

b. Western Blot Analysis

To determine the binding specificity among different species ofanti-gelsolin antibodies, western blot analysis of serum samples withanti-gelsolin antibodies was performed. The serum samples of selectmammalian species (20 μl of 1:20 dilution of serum in PBS) werefractionated by 10% SDS-PAGE and western blotted onto nitrocellulosemembrane using standard techniques. After electrotransfer the withelectroblotted nitrocellulose membrane (blot) was blocked using 5% (w/v)non-fat milk at room temperature for 1 h. Blots was probed with apurified anti-gelsolin antibody (1 μg/ml; Protein A or protein Gpurified), i.e., GN3E9; GC1C10; GF2D6; or GS 2C4, at room temperaturefor 2 h. Unbound anti-gelsolin antibody was rinsed from the blots bywashing five (5) times with PBS containing 0.02% (v/v) Tween 20 at roomtemperature for 10 min with shaking. The bound anti-gelsolin antibodywas detected by probing each blot with HRP-conjugated goat anti-murineIgG (1:10,000 in blocking buffer) at room temperature for 1 h. UnboundHRP-conjugated goat anti-murine IgG was rinsed from the blots by washingfive (5) times with PBS containing 0.02% (v/v) Tween 20 at roomtemperature for 10 min with shaking. The anti-gelsolin-HRP-conjugatedgoat anti-murine IgG complexes were visualized using BRP-mediatedchemiluminescence. Specifically, the blots were incubated with LumiGLO®Peroxidase Chemiluminescent Substrate (KPL, Gaithersburg, Md.) for 3min, and exposed to X-ray films. Results of western blot analysis areshown in FIG. 5A (GC1C10), FIG. 5B (GF2D6) and FIG. 5C (GN3E9). Thelanes were as follows: M: molecular weight marker; B: blank; lane 1:mouse serum, lane 2: monkey serum, lane 3: rabbit serum, lane 4: ratserum, lane 5: bovine serum, lane 6: horse serum, lane 7: human serum.

The results of studies examining crossreactivity of anti-gelsolinantibodies are summarized in Table 12, where “nd” is not determined.

TABLE 12 Crossreactivity of Antibodies pGSN Clone human monkey bovinehorse pig rabbit rat mouse GN3E9 + + − − nd − − − GC1C10 + + + + nd − +− GF2D6 + + + + nd − − − GS2C4 + nd + nd + + − −

As shown in Table 12, each anti-gelsolin antibody tested had a distinctpattern of crossreactivity for immunoreactive gelsolin. For example,antibody GN3E9 selectively bound primate serum gelsolin (human andmonkey), but not serum gelsolin polypeptide of the other mammals tested.In contrast, commercial anti-gelsolin antibody GS2C4 was observed tobind to most of the mammalian serum gelsolin tested, except for rat andmouse.

c. Sequence Homology Analysis of Epitopes.

Differences in the sequence alignment of 50 amino acid gelsolin epitoperegions (identified above) for various mammalian species examined forcross-reactivity with the human anti-gelsolin antibodies (see Table 12)allowed for further refinement of the gelsolin epitopes to approximately10 amino acid residues for each of the anti-gelsolin antibodies tested.The results of homology search and epitope determination are summarizedin Table 13 (GN3E9), Table 14 (GF2D6), Table 15 (GC1C10) and Table 16(Sigma GS2C4). It was observed that one or two amino acid residuechanges in the gelsolin epitope regions may affect epitope binding ofanti-gelsolin antibody.

TABLE 13 Homology search and epitope determination of GN3E9 SequenceDetermined amino acid Species homology Reactivity epitope range Seq IDHuman FAQGAL K SED ++++ FAQGAL K SED 321-330 SEQ ID NO.: 2 BovineFAQGALRSED − SEQ ID NO.: 37 Horse FAQGALRSED − SEQ ID NO.: 37 MouseFAQGALRSED − SEQ ID NO.: 37 Rat FAQGALRSED − SEQ ID NO.: 37

As summarized in Table 13, the anti-gelsolin antibody GN3E9 is predictedto bind an epitope comprising the amino acid sequence FAQGALKSED (SEQ IDNO.: 2).

TABLE 14 Homology search and epitope determination of GF2D6 SequenceDetermined amino acid Species homology Reactivity epitope range Seq IDHuman ACSN K IGRFV ++++ ACSN K IGRFV 661-670 SEQ ID NO.: 4 Bovine ACSN KIGRFV ++++ SEQ ID NO.: 4 Horse ACSN K IGRFV ++++ SEQ ID NO.: 4 MouseACSNRIGRFV − SEQ ID NO.: 38 Rat ACSNRIGRFV − SEQ ID NO.: 38 pig ACSN KIGRFV nd SEQ ID NO.: 4

As summarized in Table 14, the anti-gelsolin antibody GF2D6 is predictedto bind an epitope comprising the amino acid sequence ACSNKIGRFV (SEQ IDNO.: 4).

TABLE 15 Homology search and epitope determination of GC1C10 SequenceDetermined amino acid Species homology Reactivity epitope range Seq IDHuman SEPD G FWEAL ++++ SEPD G FWEAL 636-645 SEQ ID NO.: 3 Bovine SEPD SFWEAL ++++ SEPD S FWEAL 636-645 SEQ ID NO.: 39 Horse SEPD S FWEAL ++++SEQ ID NO.: 39 Mouse SEPDAFWEAL − SEQ ID NO.: 40 Rat SEPD G FWEAL ++++SEQ ID NO.: 3 Pig SEPDSFWEAL nd SEQ ID NO.: 39

As summarized in Table 15, the anti-gelsolin antibody GC1C10 ispredicted to bind an epitope comprising the amino acid sequenceSEPDXFWEAL (SEQ ID NO.: 47) wherein Xaa is G or S.

TABLE 16 Homology search and epitope determination of GS2C4 SequenceDetermined amino acid Species homology Reactivity epitope range Seq IDHuman GGK AA YRTSP ++++ GGK AA YRTSP 646-655 SEQ ID NO.: 41 Bovine GGKAA YRTSP ++++ SEQ ID NO.: 41 Horse GGKATYRTSP − SEQ ID NO.: 42 MouseGGKTAYRTSP − SEQ ID NO.: 43 Rat GGKTAYRTSP − SEQ ID NO.: 43 pig GGK AAYRTSP ++++ SEQ ID NO,: 41

As summarized in Table 16, the commercial anti-gelsolin antibody GS2C4is predicted to bind an epitope comprising the amino acid sequenceGGKAAYRTSP (SEQ ID NO.:41).

As noted above, the anti-gelsolin antibodies GN3EP, GC1C10, GF2D6 andGS2C4 display distinct biochemical characteristics. Anti-gelsolinantibodies GN3EP, GC1C10, and GF2D6 show patterns of cross-reactivitywhich are distinct from one another and which also differ from thepattern of cross-reactivity observed for the commercial anti-gelsolinantibody GS2C4. Furthermore, the anti-gelsolin antibodies GN3EP, GC1C10,and GF2D6 bind to gelsolin epitopes which are distinct from one anotherand which also differ from the gelsolin epitope bound by the commercialanti-gelsolin antibody GS2C4. This is further illustrated by analysis ofthe 3D structure of the C-terminus of gelsolin (Narayan et al., FEBSLett, 552: 82-85 (2003)). A shown in FIG. 6, mapping the location of thelocation of the gelsolin epitopes identified for anti-gelsolinantibodies GC1C10, GF2D6 and GS2C4 reveal that these agents bind todiffering regions on the surface of human gelsolin polypeptide. Theresults indicate that the epitopes recognized by all the anti-gelsolinantibodies tested are in regions predicted to be positively charged.

Example 6 Capability of Recognizing Actin-Free Plasma Gelsolin

The ability of the gelsolin binding agents of the present invention torecognize the unbound (active) form of plasma gelsolin and not the bound(inactive) form of plasma gelsolin was tested using an actin inhibitoryELISA assay. In this assay, an ELISA plate was coated with 1 μg/ml ofpurified native human plasma gelsolin in PBS at 4° C. overnight. Afterwashing three times with PBS, the plate was blocked with 3% (w/v) BSAPBS at room temperature for 1 h. F-actin was added to appropriate testwells to a final concentration of 10 μg/ml the presence of 10 mM CaCl₂.The plate was then incubated at incubated at 37° C. for 1 h to allowbinding of actin to the native plasma gelsolin coating on the well.Following this incubation period, the unbound F-actin was rinsed fromthe wells by washing three times with PBS at room temperature (3 mineach). Anti-gelsolin test antibody (at 1 μg/mL final concentration) wasadded to appropriate wells and incubated at 37° C. for 1 h. The unboundantibody was removed from the wells by washing the plate three timeswith PBS. Bound anti-gelsolin antibody was detected by incubating wells(30 min; 37° C.) with PRP-conjugated anti-mouse IgG antibody (diluted1:10,000; SouthernBiotech, Birmingham, Ala.)). Unbound HRP-conjugatedanti-mouse IgG antibody was removed by rinsing the wells three timeswith PBS (5 min each). Anti-gelsolin antibody-HRP-conjugated anti-mouseIgG antibody complexes were measured using SureBlue TMB 1-ComponentMicrowell Peroxidase Substrate (KPL, Gaithersburg, Md.). Specifically,SureBlue TMB 1-Component Microwell Peroxidase Substrate (KPL,Gaithersburg, Md.) was added to the wells and the plate was incubatedfor 10 min to allow HRP-mediated conversion of the substrate. Theenzymatic reaction was stopped with the addition of 100 μl 2N H₂SO₄. Theoptical density of the sample wells was then measured at 450 nm/650 nmusing an ELISA plate reader.

Comparison of the amount of anti-gelsolin antibody bound to the samplestreated with F-actin relative to the binding of antibody to samples nottreated with F-actin (100% binding) is summarized in FIG. 7. Thecommercial anti-gelsolin antibody GS2C4 did not discriminate betweenfree gelsolin (i.e., not bound to F-actin) and F-actin-bound gelsolin asno significant change was observed in the assay signal observed foranti-gelsolin antibody GS2C4 binding in the presence or absence ofF-actin treatment. In contrast, anti-gelsolin antibodies GN3E9, GC1C10,and GF2D6 show a preference for the active form of human plasma gelsolinover the actin-bound form of gelsolin (FIG. 7). While not wishing to belimited by theory, free and actin-complexed gelsolin molecules differ intheir functional properties. For example, although free gelsolin cansever actin filaments, actin-gelsolin in complexes cannot. Accordingly,use of such gelsolin binding agents which preferentially bind freeactive gelsolin in methods of the present invention have the advantageof more accurately quantifying level of free gelsolin in a biologicalsample which (as noted above) has different functional properties thangelsolin complexed with actin.

Example 7 Immunoassay for Plasma Gelsolin

1. Methods

To develop a sandwich gelsolin ELISA for quantitative measurement ofplasma gelsolin, four anti-gelsolin antibodies were selected todetermine their ability to serve as a capture or detection antibody.Gelsolin ELISA was carried out as follows. An ELISA plate (96 well; BDbiosciences CA) was coated with 10 μg/ml of the capture antibody at 4°C. overnight. Unbound capture antibody was rinsed from the wells bywashing the plate three times with PBS. Non-specific binding sites werethen blocked by incubating the wells with 3% (w/v) BSA in PBS (1 h, roomtemperature). Blocking solution was removed from the wells and the platewas air dried prior to vacuum sealing and storage at 4° C. prior to use.For measurement of gelsolin in biological sample, 50 μl of human plasmasample was first added to appropriate wells of gelsolin ELISA platetreated with capture antibody which had been equilibrated to roomtemperature. Immediately following the addition of the samples to theplate, 50 μl of HRP-conjugated detection antibody (˜0.1 μg/ml inblocking buffer) was added to appropriate wells and the plate wasincubated for 20 min at 37° C. Unbound material was rinsed from thewells by washing the plate three times with PBS. Captured plasmagelsolin: HRP-conjugated antibody complexes were measured by adding 100μl ECL substrate buffer (KPL, Inc., Gaithersburg, Md.) was added. Afterincubation at 37° C. for 3 min, the optical density of each well wasmeasured at 450 nm/650 nm in an ELISA plate reader.

2. Pairing Capability of Gelsolin Antibodies in ELISA

Table 17 shows the pairing compatibility of select antibodies of thepresent invention and the commercial anti-gelsolin monoclonal antibodyGS2C4. When anti-gelsolin antibody GN3E9 was used as the captureantibody, the highest OD values were obtained with either GC1C10 orGF2D6 antibodies as a detection antibody, suggesting that a usefulantibody configuration for gelsolin ELISA is GN3E9 as capture and GC1C10antibody or GF2D6 antibody as detection antibody. In contrast, thegelsolin binding agents of the invention (e.g., anti-gelsolin antibodiesGN3E9, GC1C10 or GF2D6), the commercial anti-gelsolin antibody GS2C4 wasnot useful as either a capture or detection antibody to detect plasmagelsolin in ELISA format.

TABLE 17 Pairing Capability of Gelsolin Antibodies in ELISA CaptureAntibody GN3E9 GC1C10 GF2D6 GS2C4 Detection GN3E9 — 2.58 2.96 0.015GC1C10 3.65 — 0.35 0.012 GF2D6 3.85 0.41 — 0.031 GS2C4  0.025  0.014 0.013 —

3. Quantitative Measurement of Plasma Gelsolin

To test the ability of gelsolin binding agents of the invention toquantitatively measure plasma gelsolin in a gelsolin ELISA assay format(as detailed above), standard curves were generated using a dilutionseries of samples containing affinity-purified human plasma gelsolin.The results are shown in FIG. 8 and indicate that the twocapture/detection antibody pairs tested are capable of quantitativemeasurement human gelsolin over a broad concentration range of plasmagelsolin. As shown in both FIG. 8A and FIG. 8B, there was a directlinear relationship between the assay signal expressed as relative lightunits (RLU) and the plasma gelsolin concentration (ng/ml). For example,the antibody pair shown in FIG. 8A (GN3E9 (capture)/GC1C10 (detection))had an R value of 0.9998. The antibody pair shown in FIG. 8B (GN3E9(capture)/GF2D6 (detection)) had an R value of 0.9989.

4. Effects of Sample Preparation on Measurement of Plasma Gelsolin

To test whether blood collection procedure may affect the quantitationof gelsolin, blood samples from 8 healthy individuals were collected inthe serum preparation tubes containing sodium citrate, herparin, or EDTA(Liu Yang Medical Device Co Ltd, Hunan, China) Gelsolin ELISA wasconducted as described above using the GN3E9/GC1C10 antibody pair. Theresults are shown in FIG. 9. The no additive (serum) sample showed agelsolin level of 126±14 μg/mL. Addition of heparin or EDTA did notsignificantly interfere with the quantitation of gelsolin in serum andshowed gelsolin levels of 124±18 μg/mL and 116±17 μg/mL, respectively.However, sodium citrate does interfere with the ELISA measurement ofhuman plasma gelsolin, showing 77±12 μg/mL gelsolin (p<0.0001). Theseresults indicate that the conditions of preparation of plasma samplesmay affect detection of immunoreactive gelsolin polypeptides by ELISAtechniques.

5. Specific Measurement of the Full-Length Gelsolin

To determine whether the immunoassay using gelsolin binding agentsmeasures full-length gelsolin and/or other immunoreactive fragments,four different types of human gelsolin standards (e.g., native gelsolin,recombinant full-length, recombinant N-terminal and recombinantC-terminal fragments) were measured by ELISA using GN3E9/GC1C10 antibodypair. The results are shown in FIG. 10. The gelsolin binding agentstested were useful in a ELISA format to quantitatively measure bothnative plasma gelsolin and recombinant full-length gelsolin in adose-dependent fashion. Consistent with the specificity of the GN3E9capture antibody and the GC1C10 detection antibody this antibody pairingdid detect either N-terminal or C-terminal fragment. These resultsindicate that the quantitative assay described in this example isspecific for full-length gelsolin and does not detect immunoreactivefragments.

6. Specific Measurement of Actin-free Gelsolin

An immunoassay for plasma gelsolin that measures the functional form ofgelsolin in samples, e.g. the actin-free form would be advantageous. Todetermine whether gelsolin ELISA using gelsolin binding agents of theinvention only measures actin-free gelsolin, various amounts of nativeplasma gelsolin standard were measured in the presence or absence of 10μg/ml F-actin using two pairs of antibodies. As shown in FIG. 11, thepresence of F-actin significantly reduced the reactivity of the pairedantibodies to gelsolin (Panel A: GN3F9/GC1C10; Panel B: GN3E9/GC2D6).These results indicate that the selected antibody pairs are useful toquantitate active plasma gelsolin in an ELISA format as they areselective for active plasma gelsolin.

Example 8 Quantitation of Plasma Gelsolin in Clinical Samples

1. Serum Gelsolin Levels in Critical Care Patients

To test the ability of the gelsolin ELISA assay to quantitate gelsolinin a clinical setting, samples from normal patients and ICU patientswere obtained and analyzed as described above. This analysis used thecapture/detection antibody pair GN3E9/GC1C10. The results are shown inFIG. 12 and Table 18. FIG. 12 shows that normal subjects exhibited aserum gelsolin level of 136±22 μg/mL, while ICU patients had asignificantly decreased serum gelsolin level of approximately 35±25μg/mL (p<0.0001). Table 18 presents data showing the sex, age,diagnosis, and actual gelsolin level for each of the critical carepatients. Thus, serum gelsolin level is a biomarker of septic shock,infection (e.g., pneumonia and respiratory distress syndrome); heartfailure; heart attack and pancreatitis in ICU patients.

TABLE 18 Serum Gelsolin Levels in Critical Care Patients Serum Cut-offgelsolin (mean − Sample No Sex Age Diagnosis (μg/ml) 3SD) Normal control136 ± 22 70 (298) 1 F 70 Stroke 8.4 **** 2 M 80 pneumonia 99.5 3 M 74Sepsis 25.8 *** 4 F 70 Pneumonia, RDS? 4.6 **** 5 F 88 Heart infarction16.8 *** 6 F 76 Heart failure 6.7 **** 7 M 72 Pneumonia, RDS? 15.2 *** 8M 80 Pneumonia, RDS? 8.7 **** 9 M 80 Pneumonia 42.2 ** 10 F 82 Pneumonia53.2 * 11 M 75 Acute pancreatis 85.4 12 M 72 ? 25.1 *** 13 46.1 * 1443.8 * 15 23.7 **** 16 43.3 * 17 F 87 Pneumonia, RDS? 21.1 *** 18 M 75Pneumonia 10.3 *** 19 M 80 Pneumonia 62.3 * 20 M 78 Sepsis 28.1 *** 21 M72 Heart failure 55.5 * 22 F 85 Heart failure 44.9 *

While the patients in the ICU group showed relatively advanced ages,other studies showed that plasma gelsolin levels among various agegroups are not significantly different. The results are shown in Table19.

TABLE 19 Quantitation of Serum Gelsolin In Human Subjects Classified byAge Age Group 20-30 30-40 40-50 50-60 60-70 70-80 Number of 83 36 13 2972 70 subjects Mean 185.8 181.1 190.2 188.4 194.6 203.7 Std. Deviation37.75 34.2 37.46 32.93 43.69 40.34 Std. Error 4.144 5.7 10.39 6.1145.149 4.822

Two additional groups of critical care patients, patients having majorsurgery and those exhibiting symptoms of severe sepsis, also showeddecreased levels of serum gelsolin using the gelsolin binding agents ofthe present invention in a gelsolin ELISA assay. Major surgery isdefined as any surgical procedure that involves anesthesia orrespiratory assistance. The criteria for patient recruitment waspreviously described (Wang et al., Eur J Clin Pharmacol 62:927-31(2006)). Severe sepsis is defined as sepsis associated with new organdysfunction, hypotension, or hypoperfusion. The criteria for patientrecruitment was previously described (Chen et al., Genes immun 8:439-43(2007)).

Compared to the control (127.7±35.2 μg/mL, n=14), serum gelsolin levelswere reduced in both the surgery patients (44.75±25.0 μg/mL, n=43) andsevere sepsis patients (21.65±12.04 μg/mL, n=80). These results furtherdemonstrate the ability of the gelsolin binding agents of the presentinvention to quantify serum gelsolin in a clinical setting. Further, thestudies demonstrate that gelsolin ELISA using gelsolin binding agents ofthe invention is useful to measure serum gelsolin in biological samplefrom sepsis patients. Gelsolin is a biomarker in human sepsis.

2. Serum Gelsolin Levels in Systemic Lupus Erythematosus (SLE)

Gelsolin ELISA using gelsolin binding agents of the invention was usedto examine whether serum gelsolin level provides a suitable biomarkerfor the detection of an active autoimmune disease, such as systemiclupus erythematosus (SLE). The gelsolin ELISA analysis was carried outas described above. The results are shown in FIG. 13 and indicate thatpatients having active SLE (2616 μg/mL, p<0.0001) or inactive SLE (88±36μg/mL, p<0.0001) show significantly decreased levels of serum gelsolincompared to normal patients (137±16 μg/mL). Likewise, patients havinginactive SLE show significantly decreased levels of serum gelsolincompared to normal patients. Further, the studies demonstrate thatgelsolin ELISA using gelsolin binding agents of the invention is usefulto measure serum gelsolin in biological sample from patients withautoimmune disease. Serum gelsolin may be a biomarker for an autoimmunedisease and may help to identify individuals moving from active toinactive SLE status.

3. Serum Gelsolin Levels in Chronic Hepatitis

Gelsolin ELISA using gelsolin binding agents of the invention was usedto examine whether serum gelsolin level provides a suitable biomarkerfor the detection of chronic hepatitis. The gelsolin ELISA analysis wascarried out as described above. As shown FIG. 14, serum gelsolin issignificantly decreased in patients with chronic hepatitis. Thus,gelsolin may be an indicator of liver function, and suggests thatpatients with chronic hepatitis may need gelsolin replacement therapy.Serum gelsolin may be a biomarker for chronic hepatitis. Further, thestudies demonstrate that gelsolin ELISA using gelsolin binding agents ofthe invention is useful to measure serum gelsolin in biological samplefrom patients with chronic hepatitis.

4. Serum Gelsolin Levels in Rheumatoid Arthritis

Gelsolin ELISA using gelsolin binding agents of the invention was usedto examine whether serum gelsolin level provides a suitable biomarkerfor the detection of a chronic inflammatory disease, such as rheumatoidarthritis. The gelsolin ELISA analysis was carried out as describedabove. The results are shown in FIG. 23 and indicate that patientshaving rheumatoid arthritis (140.116.3 μg/mL, p<0.0001; n=29; mean±SEM)show significantly decreased levels of serum gelsolin compared to normalpatients (231±4.7 μg/mL; n=32). The studies demonstrate that gelsolinELISA using gelsolin binding agents of the invention is useful tomeasure serum gelsolin in biological sample from patients with chronicinflammatory disease, e.g., rheumatoid arthritis.

5. Serum Gelsolin Levels in Cancer Patients

Gelsolin ELISA using gelsolin binding agents of the invention was usedto measure serum gelsolin levels in cancer patients in order to evaluatethe patient's condition. Serum samples were collected from patients withnewly diagnosed cancer prior to any major treatment (surgery,chemotherapy, or radiation therapy). The samples were analyzed bygelsolin ELISA as described above using the capture/detection antibodypair GN3EP/GC1C10. The results are shown in Table 20.

TABLE 20 Serum Gelsolin Levels in Cancer Patients Below cut-off Numberof Mean − 3SD % of patients below samples (70 μg/mL) cutoff value Normal291 0 0 Breast 48 7 14.6% Colon 66 17 27.6% Gastric 98 40 40.8% Lung 8842 47.7%

The results indicate that a significant portion of patients havingcancers of all the types tested exhibited a significantly lower serumgelsolin level compared to healthy controls. As such, serum gelsolinlevel may be a biomarker for cancer or the status of cancer patients.Further, the studies demonstrate that gelsolin ELISA using gelsolinbinding agents of the invention is useful to measure serum gelsolin inbiological sample from patients with cancer. The gelsolin ELISA usinggelsolin binding agents of the invention is useful in methods todetermine the status of cancer patients for the purpose of gelsolinreplacement therapy. For example, gelsolin level in a cancer patient maybe assessed to determine if the gelsolin level is decreased relative toa control reference standard. If the level of serum gelsolin in thepatient is lower than the control reference standard, then the patientmay be classified as an individual in need of gelsolin replacementtherapy. Further, the gelsolin binding agents of the invention may beused to determine the level of gelsolin to be dosed to the patient basedon the level of plasma gelsolin. Moreover, the gelsolin binding agentsof the invention may be used to measure the subsequent response of apatient to gelsolin replacement therapy by measuring serum gelsolinlevel after treatment of the patient.

Example 9 Effect of Chemotherapy on Plasma Gelsolin Levels

1. Effect of Chemotherapy on Plasma Gelsolin in an In Vivo Murine Model

The effects of chemotherapy on plasma gelsolin were investigated usingthe binding agents of the present invention. The chemotherapeutic agentstaxol (Taxol, Mayne Pharma Pty Ltd, Mulgrave VIC 3170 Australia) oradriamycin (Adriamycin, Ben Venue Laboratories, Inc. Bedford, Ohio 4414)were administered by i.p. injection (250 μg per dose) into mice (8week-old female Balb/c purchased from Animal Facility of Chinese Academyof Medical Sciences). The agent was administered two times every otherday. At three days after the last injection, serum was collected fromthe mice and analyzed by western blot using anti-gelsolin antibodiesGC1C10 and GC1G12 (using procedures described above). Antibody GC1G12cross-reacts with murine gelsolin. The results are shown in FIG. 15(Panels A and B). Each lane in panel A represents a different subjectmouse. The western blot data of panel A (FIG. 15A) was quantitativelymeasured by densitometry (FIG. 15B). The results indicate thatchemotherapy treatment using either taxol or adriamycin significantlydecreases the levels of detectable serum gelsolin in mice administeredsuch therapy.

To examine the time-dependent decrease of plasma gelsolin afterchemotherapy, mice (8 week-old female Balb/c purchased from AnimalFacility of Chinese Academy of Medical Sciences) were i.p. injected witha high dose of taxol (500 μg per dose) or adriamycin (500 μg per dose),as a single bolus injection. Serum levels of gelsolin were measured atthe indicated time point after treatment using western blot analysiswith anti-gelsolin antibody GC1G12 (as described above). FIG. 16A showsthe western blot, and FIG. 16B shows quantitation of the western blotusing densitometry. As shown in FIGS. 16A and 16B, a time-dependentdecrease of serum gelsolin after taxol treatment was observed. Likewise,as shown in FIGS. 16C and 16D, the time-dependent decrease of serumlevels of gelsolin after adriamycin treatment was observed. Thus,chemotherapy depletes plasma gelsolin in a murine model and plasmagelsolin levels may serve as a biomarker for the acute toxic response ofchemotherapy. As such, the gelsolin binding agents of the invention areuseful to monitor patient condition following chemotherapy or toascertain whether gelsolin replacement therapy, or further chemotherapy,might be appropriate. Further, the gelsolin binding agents of theinvention may be used to determine the level of gelsolin to be dosed tothe patient based on the level of plasma gelsolin. Moreover, thegelsolin binding agents of the invention may be used to measure thesubsequent response of a patient to gelsolin replacement therapy bymeasuring serum gelsolin level after treatment of the patient.

2. Effect of Chemotherapy on Plasma Gelsolin in Humans

Serum levels of gelsolin in five (5) human patients with ovarian cancerwere measured by ELISA using GN3E9/GC1C10 antibody pair (as describedabove) before and after chemotherapy. Specifically, human subjects withstage III/IV ovarian cancer received paclitaxel 185 mg/m2 IV over 3hours and cisplatin at a dose of 75 mg/m2 every three weeks aschemotherapy. The results are shown in FIG. 17. All patients showedsignificantly decreased levels of gelsolin at three weeks afterchemotherapy. Consistent with the results in the murine model,chemotherapy depletes plasma gelsolin in humans and plasma gelsolinlevels may serve as a biomarker for the acute toxic response tochemotherapy. As such, the gelsolin binding agents of the invention areuseful to monitor patient condition following chemotherapy or toascertain whether gelsolin replacement therapy, or further chemotherapy,might be appropriate. Further, the gelsolin binding agents of theinvention may be used to determine the level of gelsolin to be dosed tothe patient based on the level of plasma gelsolin. Moreover, thegelsolin binding agents of the invention may be used to measure thesubsequent response of a patient to gelsolin replacement therapy bymeasuring serum gelsolin level after treatment of the patient.

Example 10 Gelsolin Replacement Therapy

The effect of gelsolin replacement therapy on body weight and percentsurvival of mice (8 week-old female Balb/c purchased from AnimalFacility of Chinese Academy of Medical Sciences) following chemotherapywas examined. In this experiment, mice were administered two doses of250 μg adriamycin every other day by i.p. injection. One day followingthe last dose of adriamycin, the mice in the test group were provided asupplement of 100 μg recombinant full-length gelsolin by ip injection.The gelsolin supplements were repeated every other day for total ofthree doses. The body weight and percent survival of the mice followingchemotherapy and gelsolin replacement therapy are shown in FIG. 18. Theresults indicate that providing gelsolin results in 100% survival of themice after 10 days, whereas mice not provided gelsolin exhibit 100%mortality after 10 days. Likewise, the decrease in body weight for miceprovided gelsolin replacement therapy was not as severe as mice notprovided such therapy. Thus, supplementing subjects with gelsolin duringchemotherapy reduces chemotherapy-induced acute toxic response andmortality. As such, the gelsolin binding agents of the invention areuseful to monitor patient condition following chemotherapy or toascertain whether gelsolin replacement therapy, or further chemotherapy,might be appropriate. Further, the gelsolin binding agents of theinvention may be used to determine the level of gelsolin to be dosed tothe patient based on the level of plasma gelsolin. Moreover, thegelsolin binding agents of the invention may be used to measure thesubsequent response of a patient to gelsolin replacement therapy bymeasuring serum gelsolin level after treatment of the patient.

Example 11 Immunoaffinity Purification of Native Gelsolin from Plasma

1. Affinity-purification of Plasma Gelsolin

The ability of the selected antibodies of the present invention to bindto actin-free gelsolin in plasma suggests that these antibodies may haveutility for purification of native and functional form of gelsolin fromhuman plasma. To test this possibility, highly purified anti-gelsolinantibodies (Protein A or Protein G affinity purified by standardtechniques), GN3E9, GF2D6, or GC1C10 were immobilized to Sepharose 4Band used for affinity purification of human plasma gelsolin. Theimmobilization of anti-gelsolin antibodies to Sepharose 4B was carriedout as described in Example 3 above. For affinity purification of plasmagelsolin, 10 ml of pooled human plasma (at least 20 subjects) was passedthrough a 2 ml column containing antibody (e.g., anti-gelsolin antibody,GN3E9, GF2D6, or GC1C10)-immobilized beads at a flow rate of 2 ml perminute. After plasma samples were passed through each immunoaffinitycolumn, unbound material was washed from the columns with 50 ml PBS. Theprotein bound to each of the gelsolin affinity columns was eluted withelution buffer (0.1 M glycine (pH 2.4), 0.15 M NaCl). The opticaldensity of each eluted fraction (1 ml) was measured at OD280 nm. Thefractions having an OD280>0.1 units were collected. The pH Afteraddition of 100 μl of neutralization buffer (1M Tris-HCl pH 8.5), theeluates were placed separately in dialysis tubing, and the eluatesdialyzed against 1 L of PBS (pH 7.5) at 4° C. The dialysis buffer waschanged twice. The purified protein was concentrated to 1 mg/ml using acentricon filtration apparatus by standard technique. The concentratedsample was sterilized by passage through a 0.22 μm filter and thenstored at 4° C. until use. The protein purity was examined by 10%SDS-PAGE as summarized in FIG. 19 by procedures described above. FIG. 19shows the results of the fractionation of affinity-purified human plasmagelsolin using beads conjugated to anti-gelsolin antibodies GC1C10,GN3EP, and GN2D6, respectively, by 10% SDS-PAGE and Coomassie Bluestaining. Gels were stained with Coomassie Blue at room temperature for30 minutes and then destained with 50% (v/v) methanol and 10% (v/v)acetic acid. Consistent with the immunoprecipitation studies presentedin Example 3, anti-gelsolin antibodies GN3E9, GF2D6, or GC1C10 boundimmunoreactive polypeptide which migrated as an ˜90 kDa polypeptide onSDS-PAGE. The migration of the ˜90 kDa anti-gelsolin antibodyimmunoreactive polypeptide is consistent with the expected migration offull-length gelsolin polypeptide from human serum sample. The identityof this ˜90 kDa polypeptide has been confirmed to be full-lengthgelsolin polypeptide by mass spectroscopy analysis (See Example 4). Thepurity of the immunopurified full-length gelsolin polypeptide wasgreater than 90% as determined by densitometry analysis of the SDS-PAGEgel. As such, the gelsolin binding agents (e.g., anti-gelsolinantibodies GN3E9, GF2D6, or GC1C10) are useful in methods of purifyingnative human gelsolin from human serum. Likewise, these gelsolin bindingagents are useful in methods to purify immunoreactive gelsolin (e.g.,native gelsolin and recombinant gelsolin as well as fragments andhomologs, thereof) from a biological sample.

2. Comparison of the Biological Activity of Affinity-purified, NativeForm of Gelsolin with Recombinant Gelsolin Using Gelsolin Binding Agentsof the Invention

Native plasma gelsolin human gelsolin was immunoaffinity purified usingan anti-gelsolin antibody of the invention by procedures essentially asdescribed above (Example 11, section 1). Full-length recombinant humangelsolin was purified using a Ni-NTA superflow column (Qiagen, Valencia,Calif.) according to manufacturer's instructions (See Example 1). Thebiological activity of gelsolin test preparations was determined invitro in an F-actin severing assay. Briefly, F-actin was incubated withimmunoaffinity gelsolin (native or recombinant) at room temperature andthe proportion of actin in the supernatant (G-actin) versus the pellet(F-actin) was compared to a control reaction without gelsolin. Thebiological activity of gelsolin was defined by the amount orconcentration of gelsolin required to solubilize 50% of the F-actin in 5min. As the amount of gelsolin needed to reach this threshold increases,the less biologically active that sample of gelsolin is compared toother gelsolin samples that require a lesser amount to achieve the samelevel of actin cleavage per 5 min.

Specifically, the biological activity of affinity-purified human plasmagelsolin (native plasma gelsolin) was compared to that of recombinantfull-length human gelsolin by F-actin severing assay (Cytoskeleton Inc,Denver, Colo.). Gelsolin was diluted in the reaction buffer (50 mM Tris,pH 7.5, containing 0.1 mM CaCl₂, 0.1 mM MgCl₂, 30 mM NaCl, 1 mM DTT).F-actin substrate was prepared from rabbit muscle actin by diluting theactin in 0.5 mg/ml in general actin buffer (5 mM Tris, pH 8.0,containing 0.2 mM CaCl₂), and incubated the mixture on ice for 30 min.The mixture was clarified by centrifugation (14,000 rpm, 15 min) and thesupernatant containing G-actin was retained. One tenth ( 1/10) volume ofactin polymerization buffer (500 mM KCl containing 20 mM CaCl₂ and 10 mMATP) was then added to the supernatant and this mixture was incubated atroom temperature for 1 h to form F-actin. This F-actin preparation wasused as substrate in test reactions to determine gelsolin F-actinsevering activity. Gelsolin-mediated F-actin severing activity wasmeasured by incubating 5 μg F-actin preparation in the presence ofvarying concentrations of gelsolin test preparation (0-0.1 mg/ml) in 100μl of reaction buffer. Test mixtures were incubated at room temperaturefor 5 min and then centrifuged at 100,000×g for 1 h. The pelletscontaining F-actin were dissolved in SDS-PAGE sample buffer. Thesupernants containing G-actin were removed and precipitated with 20%TCA, and the pellets were dissolved in SDS-PAGE sample buffer. BothF-actin and G-actin were separated in 10% SDS-PAGE and stained withCoomassie Blue (as detailed above). The ratio of F-actin versus G-actinwas determined by densitometry. The results are summarized in Table 21below.

TABLE 21 F-actin severing activity of the affinity-purified gelsolin %of F-actin gelsolin 0 0.1 mg/ml 0.5 mg/ml 1.0 mg/ml Immunoaffinity- 7435 21 12 purified human native plasma gelsolin Full-length human 75 6855 19 recombinant gelsolin

As shown in Table 21, the anti-gelsolin antibody of the presentinvention is useful to purify biologically active human native plasmagelsolin using methods of the present invention. Use of the gelsolinbinding agents of the invention to purify human native plasma gelsolinis advantageous as the methods of the invention yield a purified humannative plasma gelsolin preparation with greater biological activitycompared with full-length human recombinant gelsolin purified by Ni-NTAaffinity chromatography as evidenced by F-actin severing activitysummarized in Table 21. Gelsolin preparations with greater biologicalactivity may be more efficacious when administered to a subject in needof gelsolin replacement when compared with administration of gelsolinpreparation with lower biological activity. Using gelsolin preparationsof greater biological activity may be administered at lower dosages toachieve the same therapeutic benefit to a subject. This lower dosage mayminimize potential for adverse side-effects of gelsolin replacementtherapy (e.g., immunological reaction or cytotoxicity)

The biological activity of gelsolin can also be determined by in vivoassay by evaluation of the efficacy of prevention ofchemotherapy-induced acute toxicity as described below. Mice are treatedwith sub-lethal doses of adriamycin, and injected with gelsolin. Thebody weight loss and mortality are used for evaluation of thetherapeutic efficacy of affinity-purified gelsolin. An increase in bodyweight loss or mortality in subjects receiving the gelsolin relative tosubject that do not receive gelsolin indicates that the gelsolinpreparation is biologically active.

Example 12 Characterization of 50 kDa Gelsolin-Like Polypeptide

1. Specificity of Binding Agents for Gelsolin and Gelsolin-likePolypeptides

A panel of anti-gelsolin antibodies raised against various immunogenswere tested for their ability to detect more than one immunoreactivegelsolin-like polypeptide in a western blot. Western blot analysis wascarried out as in Example 3 above. The results are shown in Table 22.Five antibodies that were obtained from mice immunized with nativeplasma gelsolin only recognize a 50 kDa gelsolin-like polypeptide (forexample, see FIG. 20B). Six of the antibodies raised against full-lengthrecombinant gelsolin only detect a 90 kDa polypeptide (for example, seeFIG. 20A) and 4 of them detect both a 90 and 50 kDa polypeptide (forexample, see FIG. 20C). The antibodies raised against the N-terminalfragment of gelsolin only react with a 90 kDa polypeptide and none ofthem recognize a 50 kDa polypeptide. The antibodies raised against theC-terminal fragment show varying reactivity profiles. Four out of twelveclones recognized a 90 kDa polypeptide, two out of 12 detect the 50 kDapolypeptide, and 6 out of 12 clones react with both of the 90 and 50 kDapolypeptides. FIG. 20 is a western blot showing the representativeresults of three different categories of antibodies with differentdetection profiles.

TABLE 22 Frequency of antibodies recognizing different forms of plasmagelsolin 90 kDa and Antibodies raised against 90 kDa only 50 kDa only 50kDa Native gelsolin (NG) 0/5  5/5  0/5  Full-length recombinant (GF)6/10 0/10 4/10 N-terminal fragment (NG) 4/4  0/4  0/4  C-terminalfragment (GC) 4/12 2/12 6/12

Gelsolin binding agents of the invention are useful in immunometricmethods (e.g., ELISA; RIA; western blot) which selectively measure fulllength gelsolin immunoreactive polypeptide and/or 50 kDa gelsolin-likeimmunoreactive polypeptide.

2. The 50 kDa Gelsolin-like Polypeptide is Present in Apoptotic Cells

It has been reported that caspase 3 cleaves gelsolin during apoptosis(Sun et al., J Biol Chem. 274: 33179-33182 (1999)). Therefore, the 50kDa gelsolin-like immunoreactive polypeptide may be derived from thecleavage of full length gelsolin by caspase 3. FIG. 21 is a western blotanalysis of cellular gelsolin with anti-gelsolin antibodies comparingcontrol and apoptotic cells. Lane 1 comprises control MIAcapa cells(pancreatic cancer cells) and Lane 2 are cells treated withCTB006-antibodies. CTB006 is a murine monoclonal antibody directed tothe TRAIL-R2 receptor. This antibody induces apoptosis of tumor cellsthat express TRAIL-R2 receptor. The results indicate that increasedlevels in intracellular gelsolin (full length, 90 kDa) are associatedwith induction of apoptosis, suggesting that gelsolin is astress-inducible protein. Moreover, a 50 kDa immunoreactive fragment isspecifically associated apoptotic cells, suggesting that the 50 kDaprotein observed in plasma may be a cleaved product of the full-lengthgelsolin.

3. Serum Gelsolin Profile in Cancer Patients

To further examine the clinical significance of the serum profile ofgelsolin, the two immunoreactive forms of gelsolin were measured in 8healthy controls (N1-N8) and 8 cancer patients (C1-C8) byimmunoprecipitation and western blot analysis using the anti-gelsolinantibody GC1C10 using procedures described above. As shown in FIG. 4,anti-gelsolin antibody GC1C10 was only able to precipitate the 90 kDaprotein. Thus, while anti-gelsolin antibody C1C10 is able to recognizethe 50 kDa immunoreactive gelsolin-like protein in a western blot, it isnot able to immunoprecipitate the shorter form (i.e., 50 kDaimmunoreactive polypeptide) of gelsolin in an immunoprecipitation assay.Western blot analysis of total serum samples shows that GC1C10 detectstwo forms of gelsolin in serum samples of healthy controls, but not incancer patients (FIG. 22), indicating that the 50 kDa form may be abiomarker for cancer or the status of cancer patients. Specifically, adecrease in the 50 kDa plasma gelsolin-like polypeptide may indicatethat a patient has cancer. Further, the studies demonstrate thatgelsolin using gelsolin binding agents of the invention is useful tomeasure serum gelsolin and gelsolin-like polypeptide in biologicalsample from patients with cancer.

Example 13 Determination of the N-Terminal Amino Acid Sequences of theHeavy and Light Chains of Anti-Gelsolin Binding Agents of the Invention

In order to obtain cDNAs of the heavy and light chains of GN3E9, GC1C10,and GF2D6, the N-terminal amino acid sequences of the heavy and lightchains of GN3E9, GC1C10, and GF2D6 can be determined by known sequencingtechniques. Five micrograms (5 μg) of the affinity-purified GN3E9,GC1C10, and GF2D6 are separated in 10% SDS-PAGE) under reducingconditions. After electrophoresis, the proteins in the gel aretransferred to a polyvinylidene difluoride membrane (“PVDF”). Aftertransfer, the PVDF membrane is washed with washing buffer 25 mM NaCl, 10mM sodium borate buffer (pH 8.0), then stained in a staining solution(50% (v/v) methanol, 20% (v/v) acetic acid and 0.05% (w/v) CoomassieBrilliant Blue) for 5 min to locate the protein bands. The PVDF membraneis then destained with 90% (v/v) aqueous methanol and the bandscorresponding to the heavy chain (the band with the lower mobility) andlight chain (the band with the higher mobility) are excised and washedwith deionized water. The N-terminal amino acid sequence of the heavyand light chains are determined by the Edman automated method using aprotein sequencer (PROCISE 491, ABI, USA). The results are summarized inTable 23 (N/A=not available).

Using the techniques described above, the following N-terminal sequenceswere determined for select gelsolin binding agents of the invention.

TABLE 23 N-terminal sequences of antibodies Clone Chains Sequence Seq IDGN3E9 Light chain DIVMTQSPATLSVTPGDR SEQ ID NO.: 44 GC1C10 Heavy chainEVQLVESGGGLVKPG SEQ ID NO.: 45 GC1C10 Light chain DVQMTSPSXLTSEQ ID NO.: 46

Equivalants

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

Other embodiments are set forth within the following claims.

1. An isolated antibody produced by a deposited hybridoma cell lineselected from the group consisting of: CGMCC Accession Nos: 2114, 2115,and 2116, or antigen binding fragments thereof.
 2. A method fordetermining presence or amount of gelsolin in a biological samplecomprising: (a) contacting a biological sample with one or moreantibodies produced by a deposited hybridoma cell line selected from thegroup consisting of: CGMCC Accession Nos: 2114, 2115, and 2116, orantigen-binding fragments thereof, under conditions wherein the one ormore antibodies or fragments thereof specifically bind to gelsolin; and(b) detecting the presence or amount of the one or more antibodies orfragments thereof bound to the gelsolin, thereby determining thepresence or amount of the gelsolin in the sample.
 3. The method of claim2, wherein the sample is contacted with the one or more antibodies orantigen-binding fragments thereof in an enzyme-linked immunosorbentassay (ELISA) comprising a detectable label.
 4. The method of claim 3,wherein the step of contacting comprises binding a first of the one ormore antibodies or fragments thereof to a substrate and contacting thesample and a second of the one or more antibodies or fragments thereofto the substrate, wherein the second antibody comprises the detectablelabel.
 5. The method of claim 4, wherein the first antibody comprisesthe antibody produced by hybridoma cell line CGMCC Accession No: 2115,or the antigen-binding fragment thereof, and the second antibodycomprises the antibody produced by the hybridoma cell line selected fromthe group consisting of CGMCC Accession No. 2114 and 2116, or theantigen-binding fragments thereof.
 6. A method for determining presenceof, or a predisposition to, a disease or condition associated withaltered levels of a gelsolin polypeptide in a first mammalian subject,the method comprising the steps of: (a) providing a test sample from thefirst mammalian subject; (b) contacting the test sample from the firstmammalian subject with one or more compounds that bind the gelsolinpolypeptide to form a compound/gelsolin polypeptide complex, whereineach of the one or more compounds is an antibody produced by a hybridomacell line selected from the group consisting of CGMCC Accession No.2114, 2115, and 2116, or antigen-binding fragments thereof; and (c)detecting a level of compound/gelsolin polypeptide complex in thecontacted test sample as indicative of a level of gelsolin polypeptidein the test sample; wherein an alteration in the level of the gelsolinpolypeptide in the test sample as compared to a reference levelindicates the presence of, or the predisposition to, the disease orcondition in the first subject.
 7. The method of claim 6, wherein thesample is contacted with the one or more antibodies or antigen-bindingfragments thereof in an enzyme-linked immunosorbent assay (ELISA)comprising a detectable label.
 8. The method of claim 7, wherein thestep of contacting comprises binding a first of the one or moreantibodies or fragments thereof to a substrate and contacting the sampleand a second of the one or more antibodies or fragments thereof to thesubstrate, wherein the second antibody comprises the detectable label.9. The method of claim 8, wherein the first antibody comprises theantibody produced by hybridoma cell line CGMCC Accession No: 2115, orthe antigen-binding fragment thereof, and the second antibody comprisesthe antibody produced by the hybridoma cell line selected from the groupconsisting of CGMCC Accession No. 2114 and 2116, or the antigen-bindingfragments thereof.
 10. The method of claim 6, wherein the disease orcondition associated with altered levels of gelsolin is selected fromthe group consisting of: septic shock, multiple organ dysfunctionsyndrome, rheumatoid arthritis, stroke, heart infarction, cancer,systemic autoimmune disease, chronic hepatitis, side-effects ofchemotherapy, and side-effects of radiation therapy.
 11. The method ofclaim 6 wherein the first mammalian subject is suspected of havingseptic shock, the reference standard comprises a control subject nothaving septic shock, and wherein a decrease in the level of gelsolinpolypeptide in the test sample of the first subject compared to thereference standard indicates that the first subject has septic shock.12. The method of claim 6 further comprising the steps of: comparing thelevel of gelsolin polypeptide in the test sample of the first subject toa reference standard level that comprises a control mammalian subjectnot having a disease or condition affecting gelsolin levels, andselecting to include the first subject with the alteration in the levelin a clinical trial.
 13. The method of claim 6 further comprising thesteps of: assigning the first subject with the altered level of gelsolinpolypeptide in the test sample to a subject class; and selecting aprophylactic or therapeutic treatment for the subject class.